WO2014161863A1 - Dermatological and/or cosmetic peptides for use in skin treatment - Google Patents

Dermatological and/or cosmetic peptides for use in skin treatment Download PDF

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Publication number
WO2014161863A1
WO2014161863A1 PCT/EP2014/056543 EP2014056543W WO2014161863A1 WO 2014161863 A1 WO2014161863 A1 WO 2014161863A1 EP 2014056543 W EP2014056543 W EP 2014056543W WO 2014161863 A1 WO2014161863 A1 WO 2014161863A1
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Prior art keywords
skin
glu
arg
acetyl
pro
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PCT/EP2014/056543
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French (fr)
Inventor
Raffaele Ingenito
Elisabetta Bianchi
Paola Pace
Annalise Di Marco
Ivan FINI
Alberto Bresciani
Sergio Altamura
Vincenzo Summa
Ralph Laufer
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Irbm Science Park S.P.A.
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Publication of WO2014161863A1 publication Critical patent/WO2014161863A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides capable of increasing the amount and/or the stability of collagen or of collagen-like molecules in eukaryote cells, in particular cells of the derm and/or epiderm, more particularly human fibroblasts.
  • the peptides of the invention can be used in a variety of dermatological applications, such as the treatment of burns, wounds, lacerations, skin ulcers, chronic wounds or bedsores. Further, peptides of the invention can be used for cosmetic treatment to improve the general state of the skin and to prevent and/or reduce signs of skin aging.
  • Skin represents the covering, or integument of the body's surface that provides protection and receives sensory stimuli from the external environment. It consists of a series of tissues of ectodermal and mesodermal origin and can have various colors, physiological and organic structure, subjected to more or less visible aging processes. As a mediator between the body and the outside world, the skin performs several functions in vertebrates. In particular, the skin has a protective function, being an anatomical barrier, it is the first line of defense against external aggression. In addition, the skin has a key role in sensitivity, through the presence of thermoreceptors, pressoreceptors and algoceptors, in thermal regulation and in controlling evaporation.
  • the skin consists of three layers of tissue: the epidermis, an outermost layer that contains the primary protective structure, the stratus corneum; the dermis, a fibrous layer of connective tissue that supports and strengthens the epidermis; and the subcutis, a subcutaneous layer of fat beneath the dermis that supplies nutrients to the other two layers and that cushions and insulate the body.
  • the skin also contains a number of elements that complement its structure and function: hair, nails and glands of various kinds.
  • Superficial epidermis is a stratified epithelium largely composed of keratinocytes that are formed by division of cells in the basal layer, and give rise to several distinguishable layers as they move outwards and progressively differentiate. Within the epidermis, there are several other cell populations, namely melanocytes, which donate pigment to the keratinocytes, Langerhans' cells, which have immunological functions and Merkel cells. The epidermis can be divided into four distinct layers: stratum basale or stratum germinativum, stratum spinosum, stratum granulosum and stratum comeum. Keratinocytes are the most abundant cell type in the epidermis.
  • the dermis is the connective tissue component of the skin, consisting of cells and extracellular matrix or ground substance.
  • Cell types include fixed cells, such as fibroblasts, mast cells, macrophages or histiocytes and fat cells, and the mobile cells originating from blood.
  • Fibroblasts are the most abundant and important cell type of the dermis, as they produce fibrous structures - collagen - and the macro molecular components of the ground substance.
  • the basis of the dermis is a supporting matrix or ground substance in which polysaccharides and protein are linked to produce macromolecules with a remarkable capacity for retaining water.
  • this matrix Within and associated with this matrix are two kinds of proteins: collagen, which has great tensile strength and forms the major constituent of the dermis, and elastin, which makes up only a small proportion of the bulk.
  • the cellular constituents of the dermis include fibroblasts, mast cells and histiocytes (monocyte/macrophages).
  • the dermis has a very rich blood supply, although no vessels pass through the dermal-epidermal junction
  • the extracellular matrix consists of fibers and amorphous component.
  • the fibers form the support structure of the dermis and ensure dermis tension/traction resistance and flexibility.
  • the extracellular matrix is the largest component of normal skin and gives the skin its unique properties of elasticity, tensile strength and compressibility.
  • Collagen is the major insoluble fibrous protein in the extracellular matrix and in connective tissue, accounting for about 75% of the weight of dry dermis.
  • fibers are composed of collagen, a fibrous protein whose synthesis begins in fibroblasts and is completed in the extracellular matrix.
  • the collagen fibrous component is poorly extensible; however, it is extremely tough and especially resistant to tension parallel to the fibers. This characteristic is important in maintaining the dynamic strength of the skin.
  • Collagen fiber molecules are produced in the rough endoplasmic reticulum of fibroblasts. Helical procollagens with three alpha-c ai are secreted and the molecular ends are cut by procollagen peptidase to become tropocollagens. The molecules are crosslinked to each other with a regular gap that forms the striped collagen fiber. The collagen molecule is formed by said tropocollagen simple protein units, organized in three amino acid chains, forming a triple spiral shaped rope: this confers rigidity and strength to the fibers, thus responsible for the function of supporting and holding the dermis.
  • the elementary units are associated with each other in the intercellular spaces to form filamentous structures even more complex named microfibrils and fibrils, which in turn are organized in a three-dimensional network and take the name of reticular fibers.
  • microfibrils and fibrils which in turn are organized in a three-dimensional network and take the name of reticular fibers.
  • Collagen is a family of inert proteins but characterized by a continuous turnover. The fibrous organization of dermal proteins and the type of molecular interactions determine the biomechanical properties (elasticity, resistance) of the skin.
  • Nonspecific proteases transform the collagen polymer in monomers, whose degradation is due to a specific collagenase. Proteolytic degradation of collagens by proteases produces small fragments which are released systemically. Degradation products of collagen are further cleaved by specific proteases, namely cathepsins, and subsequently phagocytosed by macrophages. Chronologically, aged skin shows decreased production of new collagen and increased proteolytic activity resulting in increased collagen degradation. Senescent fibroblasts show decreased synthesis of type I collagen and proliferate at a much slower rate than fibroblasts in young skin.
  • Skin aging is a process influenced by extrinsic and intrinsic factors and is manifested by a progressive loss of skin tissue, gradual loss of skin elasticity and the appearance of fine lines and wrinkles.
  • Intrinsic factors include the decrease in cutaneous blood flow, a loss of cells and cell function, the continuous generation of reactive oxygen species (ROS) during cellular metabolism and genetic predispositions.
  • Extrinsic factors include exposure to UV-light, environmental pollution, cigarette smoke and extreme weather conditions.
  • the cicatrization is the process of reconstruction of the cutaneous tissue, damaged due to an injury of any nature.
  • Structural reconstruction of dermal elastic fibers is an important action against skin aging. Moreover, the aging and wound healing are linked processes as they both share common molecular targets.
  • Wound healing has three principal phases: inflammatory, proliferative, and remodeling.
  • the inflammatory phase begins at the time of injury.
  • fibroblasts proliferate to become the dominant cell of the proliferative phase. They produce collagen, which provides structure to the wound and replaces the fibronectin-fibrin matrix.
  • Angiogenesis of new capillaries occurs to sustain the fibroblast proliferation.
  • Keratinocytes also epithelialize the wound.
  • the remodeling phase begins at about 2 to 3 weeks and can last up to 2 years. At this time, collagen synthesis and degradation reach equilibrium.
  • the correction of skin aging and dermal cicatrization -share the same sites of action in the dermis. The cicatrization is then the result of the repair of a skin tissue.
  • the signs of aging - can be considered as long-term skin lesions that require a real tissue reconstruction, through a process of cicatrization able to act on the mechanisms of collagen synthesis and on their organization. It is known that natural peptides play an important role in coordinating the biochemical processes and are able to stimulate the synthesis of collagen. For this reason, many peptides are important ingredients for the preparation of agents that can improve the general state of the skin.
  • peptides that either stimulate collagen production or down regulate collagenase activity could cosmetically benefit aging skin.
  • These peptides are small- sequence amino-acid chains that may stimulate angiogenesis, production of granulation tissue, and new collagen synthesis.
  • Three classes of peptides have been designed with these goals in mind: a) signal peptides, triggering wound healing mechanisms that activate fibroblasts; b) carrier peptides, capable of delivering copper into skin resulting in activation of enzymatic wound healing pathways; c) neurotransmitter-inhibiting peptides, interfering with stabilization step in neurotransmitter release.
  • matrikines are indicated peptides obtained by (partial) proteolytic cleavage of constituents of the extracellular matrix that are able to regulate positively or negatively cell activities and thus have activities similar to cytokines or regulatory peptide factors.
  • This class of mediators plays a significant role in physiological or pathological processes (like angiogenesis, wound healing, tumor invasion) by modulating, for example, chemotaxis, mitogenesis, and responses to stimuli causing cell death.
  • Matrikines are important components of a broad cell and tissue sensory system to detect and respond to various types of tissue injury (Maquart FX et al Biochimie. 2005; 87, 3-4, 353-60).
  • WO2000/015188 relates to the use of peptides of formula X-Thr-Thr-Lys-Y in cosmetic or dermopharmaceutical compositions. Such peptides are chemically modified to increase their lipophilicity by grafting on the N-terminal amine of X either a fatty acid chain or by esterification or amidation of the C-terminal carboxyl group of Y.
  • WO91/12014 describes the biological activity of the copper complex of the tripeptide Gly-His-Lys (GHK-Cu).
  • GHK-Cu Gly-His-Lys
  • This peptide increases fibroblastic collagen synthesis thereby enabling a more rapid replacement of the transplants with human tissue. It is present in plasma, saliva and urine in physiologically active amount and it has two main functions: (1) first as a potent tissue protective, anti-inflammatory agent that limits oxidative damage after tissue injury, and (2) as a signal that activates tissue remodeling, consisting of removal of damaged protein and scar tissue and replacement by normal tissue.
  • GHK-Cu links the processes of removal of damaged scar tissue and deposition of new tissue (Methods in Enzymology, Vol 147, 1987, pp 314-328, Academic Press).
  • WOOl/43701 relates to the use of N-palmytoyl-Gly-His-Lys (Palm-GHK) tripeptide in cosmetic or dermopharmaceutical composition for preventing or reducing the signs of skin aging.
  • Palm-GHK N-palmytoyl-Gly-His-Lys
  • W099/12968 and WO00/42071 disclose tripeptides of formula KXK, with X one of the twenty naturally occurring amino acids, and their use in therapeutic methods to inhibit or augment inflammatory response.
  • WO 2010/082177 proposes other peptidic compounds of formula KXK for the cosmetic and dermopharmaceutic fields, that are able to improve the general state of the skin and its appendages.
  • Peptides for use in the treatment and prophylaxis of inflammation of epithelial tissues and their use also for non therapeutic skin care are described in WO 2012/107244.
  • WO00/43417 discloses the use of certain peptides and derivatives as cosmetics or pharmaceutical compositions for the regulation of impaired immunologic functions and inflammations of the skin.
  • Exemplified peptides include N-Palm-Gly-Gln-Pro-Arg (Palm-GQPR).
  • Palm-GQPR N-Palm-Gly-Gln-Pro-Arg
  • the combination of Palm- GQPR and Palm-GHK is produced by Sederma under the trademark MatrixylTM 3000. The synergistic effect of the two peptides is described in WO2005/048968.
  • hAng Human angiogenin
  • pancreatic ribonuclease superfamily which is normally present in the plasma. This protein promotes the formation of new blood vessels from pre-existing blood vessels through a process called angiogenesis.
  • angiogenin is induced during inflammation, exhibiting wound healing properties as well as microbiocidal activity and conferring host immunity (Tello-Montoliu A et al, J. Thromb. Haemost. 2006, 4, 1864-1874).
  • EP622071 describes a whitening cosmetic preparation containing angiogenin for preventing the developments of blotches and freckles of skin through the inhibition of melanin production.
  • JP2004331566 describes a skin collagen production enhancer comprising angiogenin and/or an angiogenin degradate obtained by decomposing angiogenin with a protease such as pepsin or pancreatin. It is further described a drink preparation for enhancing collagen production, comprising angiogenin obtained from skim milk, useful for preventing skin roughness, wrinkles, decreased elasticity, etc.
  • peptide Ac-PHREN-NH 2 and structurally related peptides comprising from four to six aminoacids are effective in increasing amount and/or stability of collagen in the skin.
  • peptides of the invention promotes the production of collagen type I in human fibroblasts cell cultures, and are not toxic for the cells when tested at high concentration.
  • Such compounds are therefore particularly useful in the cosmetic and dermopharmaceutic fields, to improve the general state of the skin and to treat conditions ameliorated by an increased production and/or stability of collagen. Therefore, compounds of the invention can be used to prepare topical dermopharmaceutical and/or cosmetic compositions useful for facilitating wound healing, treatment of minor burns, and to prevent signs of cutaneous aging and photoaging.
  • a novel class of peptides is capable of increasing in a substantial significant way the synthesis of collagen type I in human fibroblasts cell cultures.
  • the peptides of the invention do not show cellular toxicity.
  • the newly formed collagen fibers are very important for accelerating the process of cutaneous regeneration.
  • n, p, q 0, 1 and if one of m, n, p o q is 0, the others are 1 ;
  • R 2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
  • X is selected from an amino acid, an amino acid derivative, OR 3 , NR 3 R 4 wherein R 3 and R 4 are each independently selected from H, C 1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof, with the exclusion of the compounds wherein:
  • A is selected from acetyl and palmitoyl.
  • peptide of general formula (I) is selected in the group consisting of:
  • n, p, q 0, 1 e and if one of m, n, p o q is 0, the others are 1 ;
  • R 2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
  • X is selected from a amino acid, an amino acid derivative, OR 3 , NR3R4 wherein R3 and R4 are each independently selected from H, C 1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof,
  • A is selected from acetyl and palmitoyl.
  • the peptides of the invention are also for use in the treatment of skin conditions which are ameliorated by an increase of the stability of collagen.
  • the peptides of the invention are also for use in the treatment of skin conditions associated with loss of collagen.
  • treatment comprises both a therapeutic and a preventive affect.
  • the peptide of general formula (I) for use as indicated above is selected in the group consisting of:
  • composition comprising at least one peptide of general formula (I) and a dermatologically acceptable vehicle.
  • the peptide of general formula (I) is present in said composition at a concentration between 0.0005% w/w and 0.5% w/w.
  • said composition comprises at least one additional ingredient selected from the group comprising vitamin C, derivatives of vitamin C, vitamin E, derivatives of vitamin E, tocopherols, vitamin A, derivatives of vitamin A, retinal, retinoic acid.
  • the composition further contains at least one other active ingredient.
  • the at least one other active ingredient is selected from the group consisting of: an anti-radical agent or antioxidant.
  • the composition is in the form of a cream, emulsion, dispersion, gel, ointment, lotion.
  • said composition is for use in the treatment of skin conditions which are ameliorated by an increase of the amount of collagen. It is a further object of the invention the use of said composition to prevent and/or reduce the cutaneous signs of aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress.
  • It is a further object of the invention a method for the treatment and/or prevention of a skin condition that is ameliorated by an increase of the amount and/or stability of collagen comprising at least one step that consists in applying, to the skin or scalp, at least one peptide as defined above or at least one composition as defined above.
  • peptide or “polypeptide” are not particularly restricted, and in generale designate natural or synthetic peptides containing only natural amino acids, only non-natural amino acids, or combinations of natural and non-natural amino acids.
  • peptide denotes a chain of amino acids linked together via a peptide bond (or amide bond).
  • amino acid as employed herein includes and encompasses all of the naturally occurring amino acids, either in the D-, L-, alio, or other stereoisomeric configurations if optically active, as well as any known or conceivable non-natural, synthetic and modified amino acid.
  • natural amino acids denotes the following 20 amino acids in their laevorotatory (L) or dextrorotatory (D) form, preferably in their natural L form:
  • ydrophobic amino acid denotes one of the following amino acids: I, L, V, M, F, Y, W, T, G, C or A.
  • alkaline hydrophilic amino acid denotes one of the following amino acids: R, K or H.
  • neutral hydrophilic amino acid denotes one of the following amino acids: S,P, N or Q.
  • acidic amino acid denotes one of the following amino acids: D or E.
  • polypeptides as employed herein includes and encompasses oligopeptides, peptides, polypeptides and derivatives thereof, peptide analogs and derivatives thereof, as well as pharmaceutically acceptable salts of said compounds.
  • peptides as employed herein includes complexes with other species, such as metal ions (like copper, zinc, manganese, magnesium etc.). According to preferred embodiments of the invention, particularly interesting are the complexes with copper ions as the peptide of sequence AC-PHREN-NH 2 represents the aminoacidic sequence involved in the binding of angiogenin to copper(II).
  • hexapeptide indicates compounds including a sequence of, respectively, six, five and four amino acids in consecutive order. These amino acids are indicated using the three or one letter codes, according to international conventions, from the N- terminus to the C-terminus. According to said conventions, proline is indicated as Pro or P, histidine is indicated as His or H, arginine as Arg or R, glutamic acis as Glu or E, asparagine as Asn or N, lysine as Lys or K, glutamine as Gin or Q, aspartic acid as Asp or D.
  • the non natural amino acids include, without limitations, the hydroxyproline (Hyp), the L-l,2,3,4-tetrahydroisoquinolin-3-carboxylic acid (Tic), azetidine, D-proline (pro), homo-proline (hPro), thienylalanine (Tlia), tiazolidinalanine (Thz), ornitine (Orn), nor-arginine (Agb).
  • Peptides of the invention includes, without limitations: Ac-Pro-His-Arg-Glu-Asn, Ac-Pro-His-Arg- Glu-Asn-Lys-N E Palm, Palm-Pro-His-Arg-Glu-Asn, Ac-Pro-His-Arg-Glu-Gln, Ac-Pro-His-Arg- Glu, His-Arg-Glu-Asn, Ac-Pro-Arg-Glu-Asn, Ac-Hyp-His-Arg-Glu-Asn, Ac-Pro-His-Glu-Asn.
  • lipophilicity or lipophilic character can be increased through acylation of the N- terminal amino group of the peptide, or through esterification of the carboxy terminal with an alcohol, linear or branched, saturated or unsaturated, hydroxylated or not, or through both said chemical modifications.
  • N-acyl groups are acetyl, lauroyl, miristoyl, palmitoyl, steroyl, oleoyl, lineoyl. Particularly preferred are the groups N-acetyl and N-palmitoyl.
  • Ac indicates an N-acyl derivative (indicated also as acyl-derivative).
  • Palm indicates a N-Palmitoyl derivative.
  • OAlk indicates an ester group attached to the C-terminus carboxylic group.
  • polypeptides of the invention can be obtained from chemical or enzymatic synthesis starting from the constitutive amino acids or from their derivatives; alternatively, they can be obtained from natural proteins by hydrolysis under mild conditions, or by biotechnology.
  • known methods of peptide synthesis can be applied, as the Fmoc/tBu method in solid phase.
  • Other chemical methods include Boc/bzl or liquid phase synthesis. References for the synthetic methodologies are described for example in: Solid Phase Peptide Synthesis (1984), Pierce Chemical Company, Rockford, Illinois; The Practice of Peptide Synthesis (1984), Springer Verlach, New York; Chemical Approaches to the Synthesis of Peptides and Proteins (1997), CRC, Boca Raton, FL; J. Biol. Chem. (1980), 255, 8234-8238.
  • polypeptides of the invention can form homogeneous or mixed salts with mono- or polivalent acids, preferably with inorganic acids or with appropriate aliphatic carboxylic acids saturated or unsaturated, or with aromatic carboxylic acids, or with aliphatic or aromatic solfonic acids, preferably acetic acid, lactic acid and/or chloridric acid.
  • the compounds of the invention stimulate metabolic functions of skin cells.
  • the compounds of the invention increase the amounts of the proteins constituent of the extracellular matrix, probably by increasing their synthesis.
  • compounds of general formula (I) are capable of increasing the amount of collagen type (I) in human fibroblast cell cultures and have a positive effect on tissue regeneration.
  • the peptides of the invention find use as active ingredients for the preparation of compositions preferably for dermatologic and/or cosmetic use.
  • Said compositions can be used for example to prevent or reduce signs of skin aging, in particular wrinkles, and/or to ameliorate skin appearance.
  • the use of at least one peptide in accordance with the present invention in cosmetic, personal care or dermopharmaceutical compositions has anti-aging activity.
  • Anti- aging activity means some degree or capacity for treating, preventing or ameliorating one or more signs, symptoms, and/or causes of skin aging.
  • the present peptides retard the deleterious effects of natural aging, free radical activity, and sunlight on the complexion of the skin.
  • Another embodiment of the invention consists of the use of the peptides or of a composition comprising at least one peptide to prevent or treat the cutaneous signs of aging.
  • the "cutaneous signs of aging” include, but are not limited to, any visible manifestations on the skin caused by aging. In particular, this means wrinkles, fine lines, chapped skin, enlarged pores, imperfections, losses in firmness, discoloration, aged areas, keratosis, losses in collagen, and other changes to the dermis and epidermis, etc.
  • “Cutaneous signs of aging” also means any changes to the outer appearance of the skin and skin appendages caused by aging, such as superficial roughness of the corneal layer, fine lines and wrinkles, but also any internal change to the skin which is not translated systematically into a modified outer appearance, such as thinning of the dermis or any other internal degradation of the skin following exposure to ultraviolet (UV) radiation. More specifically, the invention relates to the use of the composition as described above in order to reduce the signs of skin fatigue.
  • the use as anti-aging is for preventing and/or treating aged or senescent skins, and/or for preventing and/or treating wrinkles and/or fine lines and/or cracks, thinning of the skin, in particular of the dermis, and/or senescence spots, and/or for combating skin disorders induced by an oxidative stress chosen from a dull appearance of the complexion, and/or hyperpigmentation of the skin, and/or a loss of quality of the sebum, and/or a dandruff appearance of the scalp, and/or a feeling of discomfort on the scalp.
  • the present invention provides peptides and compositions for topical application which comprise an effective amount of a peptide of the invention to treat, reverse, ameliorate and/or prevent signs of skin aging.
  • signs of skin aging include without limitation, the following:
  • compositions of the invention are applied to skin in need of treatment. That is, skin which suffers from a deficiency or loss in any of the foregoing attributes or which would otherwise benefit from improvement in any of the foregoing skin attributes.
  • compositions and methods of the invention are directed to the prevention, treatment, and/or amelioration of fine lines and/or wrinkles in the skin.
  • the compositions are applied to skin in need of treatment, by which is meant skin having wrinkles and/or fine lines.
  • the compositions are applied directly to the fine lines and/or wrinkles.
  • the compositions and methods are suitable for treating fine lines and/or wrinkles on any surface of the skin, including without limitation, the skin of the face, neck, and/or hands.
  • the cosmetic compositions for treating a skin condition associated with loss of collagen and/or elastin fiber comprise, in a cosmetically acceptable vehicle, an amount of a peptide of the invention effective to enhance collagen.
  • These collagen enhancing/stimulating agents may have the structure of formula (I) as indicated above.
  • the compounds of the invention and the compositions comprising said compounds are useful in the treatment of skin conditions which are ameliorated by an increased amount and/or stability of collagen and/or to accelerate recovery in said skin conditions.
  • skin conditions which are ameliorated by an increased amount of collagen or skin conditions associated with loss of collagen and/or loss of collagen stability include skin wounds, therefore peptide of the inventions are useful in accelerating wound healing and in the treatment of disorders of wound healing.
  • said skin conditions also include bedsores, ulcers, in particular in the elderly and debilitated, in diabetics, in patients affected by vasculopathies and coagulopathies, in patients under glucocorticoid or immunosuppressant therapies, or showing vitamin C deficiency.
  • the general symptoms of cutaneous aging are also included within the meaning of skin conditions as used herein.
  • compositions of the invention comprise one or more peptides of general formula (I) in combination with dermatologically acceptable components.
  • compositions or the ingredients are suitable for topical use on human skin without risks of toxicity, incompatibility, instability, intolerance, allergic response and the like.
  • cosmetically or dermatologically acceptable also means compatible with the skin and/or its integuments, having a pleasant colour, odour and feel and not causing any unacceptable discomfort (stinging, tautness or redness) liable to discourage the consumer from using this composition.
  • Peptides of general formula (I) are formulated in compositions containing a dermatologically acceptable carrier.
  • the dermatologically acceptable carrier include be aqueous or hydroalcoholic solution, water in oil emulsion, oil in water emulsion, microemulsion, aqueous gel, anhydrous gel, serum or vesicle dispersion.
  • the composition of the invention may include various other and additional ingredients, which may be active, functional, conventionally used in cosmetic, personal care or topical/transdermal pharmaceutical products or otherwise. A decision to include an additional ingredient and the choice of specific additional ingredients depends on the specific application and product formulation.
  • CTFA Cosmetic Ingredient Handbook 10 th edition, 2004 (published by Cosmetic, Toiletry and Fragrance Association, Inc, Washinghton, D.C.) describes a wide variety of non limiting materials that can be added to the composition herein.
  • actives include, but are not limited to, sugar amines, vitamin B3 and its derivatives, dehydroacetic acid (DHA) and its salts, phytosterols, salicylic acid compounds, ascorbic acid and derivatives, UV absorbers, keratinolytic agents, anti-oxidants, free radical scavengers, antimicrobial agents, antibacterial agents, antifungal agents, thickeners, antiperspirants and lipid thickeners.
  • compositions of the invention comprise at least one dermato logically acceptable vehicle, selected on the basis of the final formulation of the product.
  • Said vehicle is present at a concentration comprised between 50% and 99.99%, preferably between 60% and 99.9%, more preferably between 70% and 98% by weight of the composition.
  • compositions of the invention are generally prepared with conventional methods known in the art for the preparations for dermatological and/or cosmetic use.
  • Said compositions can be formulated in different physical forms, including creams, lotions, ointments, milk, gel, emulsions, suspensions, anhydrous preparations, shampoos and scalp treatment lotions, creams or lotions for care of skin or hair, sunscreens, transdermal patches, sprays, soaps, lipsticks or, in general, products for make-up.
  • the invention embraces the use of cosmetically acceptable (e.g., non-toxic and/or non-irritating) salts.
  • the salts of the compounds in the present invention include salts with alkali metals such as sodium and potassium; salts with alkaline-earth metals such as calcium and magnesium; salts with amines such as monoethanolamine; salts with inorganic acids such as hydrochloric acid and sulfuric acid; and salts with organic acids such as citric acid and acetic acid. Special mention may be made of hydrochloride salts.
  • compositions according to the invention can be formulated in a variety of forms for topical application and will comprise from about 0.00001% to about 90% by weight of one or more compounds according to formula (I), and preferably will comprise from about 0.001% to about 25% by weight, and more preferably from about 0.001% to about 1% by weight.
  • the compositions will comprise a dermatologically effective amount of the peptide of the invention, by which is meant an amount sufficient to enhance the aesthetic appearance of a given area of skin when topically applied thereto.
  • the composition may be formulated in a variety of product forms, such as, for example, a lotion, cream, serum, spray, aerosol, cake, ointment, essence, gel, paste, patch, pencil, towelette, mask, stick, foam, elixir, concentrate, and the like, particularly for topical administration.
  • a lotion, cream, serum, spray, aerosol, cake, ointment, essence, gel, paste, patch, pencil, towelette, mask, stick, foam, elixir, concentrate, and the like particularly for topical administration.
  • the composition is formulated as a lotion, cream, ointment, or gel.
  • compositions can include a cosmetically acceptable vehicle.
  • vehicles may take the form of any known in the art suitable for application to skin and may include, but are not limited to, water; vegetable oils; mineral oils; esters such as octal palmitate, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether and dimethyl isosorbide; alcohols such as ethanol and isopropanol; fatty alcohols such as cetyl alcohol, cetearyl alcohol, stearyl alcohol and biphenyl alcohol; isoparaffins such as isooctane, isododecane and hexadecane; silicone oils such as cyclomethicone, dimethicone, dimethicone cross-polymer, polysiloxanes and their derivatives, preferably organomodified derivatives; hydrocarbon oils such as mineral oil, petrolatum, isoeicosane and polyisobutene; polyols such as propylene glycol
  • the vehicle may comprise an aqueous phase, an oil phase, an alcohol, a silicone phase or mixtures thereof.
  • the cosmetically acceptable vehicle may also comprise an emulsion.
  • suitable emulsions include water-in-oil emulsions, oil-in-water emulsions, silicone-in- water emulsions, water-in-silicone emulsions, wax-in- water emulsions, water-oil-water triple emulsions or the like having the appearance of a cream, gel or microemulsions.
  • the emulsion may include an emulsifier, such as a nonionic, anionic or amphoteric surfactant.
  • the oil phase of the emulsion preferably has one or more organic compounds, including emollients; humectants (such as propylene glycol and glycerin); other water- dispersible or water-soluble components including thickeners such as veegum or hydroxyalkyl cellulose; gelling agents, such as high MW polyacrylic acid, i.e. CARBOPOL 934; and mixtures thereof.
  • the emulsion may have one or more emulsifiers capable of emulsifying the various components present in the composition.
  • the compounds suitable for use in the oil phase include without limitation, vegetable oils; esters such as octyl palmitate, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether; fatty alcohols such as cetyl alcohol, stearyl alcohol and behenyl alcohol; isoparaffins such as isooctane, isododecane and isohexadecane; silicone oils such as dimethicones, cyclic silicones, and polysiloxanes; hydrocarbon oils such as mineral oil, petrolatum, isoeicosane and polyisobutene; natural or synthetic waxes; and the like. Suitable hydrophobic hydrocarbon oils may be saturated or unsaturated, have an aliphatic character and be straight or branched chained or contain alicyclic or aromatic rings.
  • the oil-containing phase may be composed of a singular oil or mixtures of different oils.
  • Hydrocarbon oils include those having 6-20 carbon atoms, more preferably 10-16 carbon atoms.
  • Representative hydrocarbons include decane, dodecane, tetradecane, tridecane, and C 6 -20- isoparaffins.
  • Paraffinic hydrocarbons are available from Exxon under the ISOPARS trademark, and from the Permethyl Corporation.
  • C 6 -2o- paraffinic hydrocarbons such as CI 2 isoparaffin (isododecane) manufactured by the Permethyl Corporation having the tradename Permethyl 99ATM are also contemplated to be suitable.
  • CI 2 isoparaffin isododecane
  • Permethyl 99ATM Various commercially available C 16 isoparaffins, such as isohexadecane (having the tradename Permethyl RTM) are also suitable.
  • Examples of preferred volatile hydrocarbons include polydecanes such as isododecane and isodecane, including for example, Permethyl- 99 A (Presperse Inc.) and the C7-C8 through CI 2- C15 isoparaffins such as the Isopar Series available from Exxon Chemicals.
  • a representative hydrocarbon solvent is isododecane.
  • the oil phase may comprise one or more waxes, including for example, rice bran wax, carnauba wax, ouricurry wax, candelilla wax, montan waxes, sugar cane waxes, ozokerite, polyethylene waxes, Fischer-Tropsch waxes, beeswax, microcrystaline wax, silicone waxes, fluorinated waxes, and any combination thereof.
  • waxes including for example, rice bran wax, carnauba wax, ouricurry wax, candelilla wax, montan waxes, sugar cane waxes, ozokerite, polyethylene waxes, Fischer-Tropsch waxes, beeswax, microcrystaline wax, silicone waxes, fluorinated waxes, and any combination thereof.
  • Non-limiting emulsifiers included emulsifying waxes, emulsifying polyhydric alcohols, polyether polyols, polyethers, mono- or di-ester of polyols, ethylene glycol mono- stearates, glycerin mono- stearates, glycerin di-stearates, silicone-containing emulsifiers, soya sterols, fatty alcohols such as cetyl alcohol, fatty acids such as stearic acid, fatty acid salts, and mixtures thereof.
  • the preferred emulsifiers include soya sterol, cetyl alcohol, stearic acid, emulsifying wax, and mixtures thereof.
  • the oil phase may comprise one or more volatile and/or non-volatile silicone oils.
  • Volatile silicones include cyclic and linear volatile dimethylsiloxane silicones.
  • the volatile silicones may include cyclodimethicones, including tetramer (D4), pentamer (D5), and hexamer (D6) cyclomethicones, or mixtures thereof.
  • Suitable dimethicones are available from Dow Corning under the name Dow Corning 200(R) Fluid and have viscosities ranging from 0.65 to 600,000 centistokes or higher.
  • Suitable non-polar, volatile liquid silicone oils are disclosed in U.S. Pat. No. 4,781 ,917, herein incorporated by reference in its entirety.
  • volatile silicone materials are described in Todd et al, "Volatile Silicone Fluids for Cosmetics", Cosmetics and Toiletries, 91 :27-32 (1976), herein incorporated by reference in its entirety.
  • Linear volatile silicones generally have a viscosity of less than about 5 centistokes at 25°C, whereas the cyclic silicones have viscosities of less than about 10 centistokes at 25°C.
  • volatile silicones of varying viscosities include Dow Corning 200, Dow Corning 244, Dow Corning 245, Dow Corning 344, and Dow Corning 345, (Dow Corning Corp.); SF-1204 and SF-1202 Silicone Fluids (G.E.
  • Linear, volatile silicones include low molecular weight polydimethylsiloxane compounds such as hexamethyldisiloxane, octamethyltrisiloxane, decamethyltetrasiloxane, and dodecamethylpentasiloxane, to name a few.
  • Non-volatile silicone oils will typically comprise polyalkylsiloxanes, polyarylsiloxanes, polyalkylarylsiloxanes, or mixtures thereof. Polydimethylsiloxanes are preferred non-volatile silicone oils.
  • the non-volatile silicone oils will typically have a viscosity from about 10 to about 60,000 centistokes at 25°C, preferably between about 10 and about 10,000 centistokes, and more preferred still between about 10 and about 500 centistokes; and a boiling point greater than 250° C at atmospheric pressure.
  • Non limiting examples include dimethyl polysiloxane (dimethicone), phenyl trimethicone, and diphenyldimethicone.
  • the volatile and non-volatile silicone oils may optionally be substituted with various functional groups such as alkyl, aryl, amine groups, vinyl, hydroxyl, haloalkyl groups, alkylaryl groups, and acrylate groups, to name a few.
  • the water-in-silicone emulsion may be emulsified with a nonionic surfactant (emulsifier) such as, for example, polydiorganosiloxane-polyoxyalkylene block copolymers, including those described in U.S. Patent No. 4, 122,029, the disclosure of which is hereby incorporated by reference.
  • a nonionic surfactant emulsifier
  • polydiorganosiloxane-polyoxyalkylene block copolymers including those described in U.S. Patent No. 4, 122,029, the disclosure of which is hereby incorporated by reference.
  • emulsifiers generally comprise a polydiorganosiloxane backbone, typically polydimethylsiloxane, having side chains comprising -(EO)m- and/or - (PO)n- groups, where EO is ethyleneoxy and PO is 1 ,2-propyleneoxy, the side chains being typically capped or terminated with hydrogen or lower alkyl groups (e.g., CI -6, typically CI -3).
  • CI -6 typically CI -3
  • Other suitable water-in-silicone emulsifiers are disclosed in U.S. Patent No. 6,685,952, the disclosure of which is hereby incorporated by reference herein.
  • water-in-silicone emulsifiers include those available from Dow Corning under the trade designations 3225C and 5225C FORMULATION AID; SILICONE SF-1528 available from General Electric; ABIL EM 90 and EM 97, available from Goldschmidt Chemical Corporation (Hopewell, VA); and the SILWET series of emulsifiers sold by OSI Specialties (Danbury, CT).
  • water-in-silicone emulsifiers include, but are not limited to, dimethicone PEG 10/15 crosspolymer, dimethicone copolyol, cetyl dimethicone copolyol, PEG- 15 lauryl dimethicone crosspolymer, laurylmethicone crosspolymer, cyclomethicone and dimethicone copolyol, dimethicone copolyol (and) caprylic/capric triglycerides, polyglyceryl-4 isostearate (and) cetyl dimethicone copolyol (and) hexyl laurate, and dimethicone copolyol (and) cyclopentasiloxane.
  • Preferred examples of water-in-silicone emulsifiers include, without limitation, PEG/PPG- 18/18 dimethicone (trade name 5225 C, Dow Corning), PEG/PPG-19/19 dimethicone (trade name BY25-337, Dow Corning), Cetyl PEG/PPG- 10/1 dimethicone (trade name Abil EM- 90, Goldschmidt Chemical Corporation), PEG-12 dimethicone (trade name SF 1288, General Electric), lauryl PEG/PPG- 18/18 methicone (trade name 5200 FORMULATION AID, Dow Corning), PEG-12 dimethicone crosspolymer (trade name 9010 and 901 1 silicone elastomer blend, Dow Corning), PEG- 10 dimethicone crosspolymer (trade name KSG-20, Shin-Etsu), and dimethicone PEG- 10/15 crosspolymer (trade name KSG-210, Shin-Etsu).
  • the water-in-silicone emulsifiers typically will be present in the composition in an amount from about 0.001% to about 10% by weight, in particular in an amount from about 0.01% to about 5% by weight, and more preferably, below 1% by weight.
  • the aqueous phase of the emulsion may include one or more additional solvents, including lower alcohols, such as ethanol, isopropanol, and the like.
  • the volatile solvent may also be a cosmetically acceptable ester such as butyl acetate or ethyl acetate; or the like.
  • the oil-containing phase will typically comprise from about 10% to about 99%, preferably from about 20% to about 85%, and more preferably from about 30% to about 70% by weight, based on the total weight of the emulsion, and the aqueous phase will typically comprise from about 1% to about 90%, preferably from about 5% to about 70%, and more preferably from about 20% to about 60% by weight of the total emulsion.
  • the aqueous phase will typically comprise from about 25% to about 100%, more typically from about 50% to about 95% by weight water.
  • the compositions may include liposomes.
  • the liposomes may comprise other additives or substances and/or may be modified to more specifically reach or remain at a site following administration. Additionally, the compositions may incorporate encapsulation and/or microencapsulation technology.
  • encapsulating materials can be selected which will release the composition upon exposure to moisture, pH change, temperature change, solubility change, or mechanical shear or rupture.
  • Suitable encapsulating materials and methods of preparing encapsulated materials are disclosed in, for example, U.S. Patent Application Publication No. 2005/0000531 to Shi; Uhlmann, et al, "Flavor encapsulation technologies: an overview including recent developments” Perfumer and Flavorist, 27, 52-61 , 2002; and "Selection of Coating and Microencapsulation Processes" by Robert E.
  • compositions incorporating encapsulation and/or microencapsulation technology may form nanoparticles.
  • nanoparticle refers to a nanometer-sized particle, having a diameter of between about 1 nanometer and about 999 nanometers; the term “nanoparticles” as used herein refers to nanometer-sized particles, nanoclusters, clusters, particles, small particles, and nanostructured materials.
  • composition may optionally comprise other cosmetic actives and excipients, obvious to those skilled in the art including, but not limited to, fillers, emulsifying agents, antioxidants, surfactants, film formers, chelating agents, gelling agents, thickeners, emollients, humectants, moisturizers, vitamins, minerals, viscosity and/or rheology modifiers, sunscreens, keratolyses, depigmenting agents, retinoids, hormonal compounds, alpha- hydroxy acids, alpha-keto acids, anti-mycobacterial agents, antifungal agents, antimicrobials, antivirals, analgesics, lipidic compounds, anti-allergenic agents, HI or H2 antihistamines, anti-inflammatory agents, anti-irritants, antineoplastics, immune system boosting agents, immune system suppressing agents, anti-acne agents, anesthetics, antiseptics, insect repellents, skin cooling compounds, skin protectants, skin penetration enhancers, ex
  • the cosmetic compositions of the invention may contain any other compound for the treatment of skin disorders.
  • additional actives and/or excipients may work in concert with the compositions of the current invention to achieve cumulative or synergistic improvements in the aesthetic appearance.
  • Colorants may include, for example, organic and inorganic pigments and pearlescent agents.
  • Suitable inorganic pigments include, but are not limited to, titanium oxide, zirconium oxide and cerium oxide, as well as zinc oxide, iron oxide, chromium oxide and ferric blue.
  • Suitable organic pigments include barium, strontium, calcium, and aluminium lakes and carbon black.
  • Suitable pearlescent agents include mica coated with titanium oxide, with iron oxide, or with natural pigment.
  • Fillers are normally present in an amount of about 0 weight % to about 20 weight %, based on the total weight of the composition, preferably about 0.1 weight % to about 10 weight %.
  • Suitable fillers include without limitation silica, treated silica, talc, zinc stearate, mica, kaolin, Nylon powders such as Orgasol(TM), polyethylene powder, Tefion(TM), starch, boron nitride, copolymer microspheres such as Expancel(TM) (Nobel Industries), Polytrap(TM) (Dow Corning) and silicone resin microbeads (Tospearl(TM) from Toshiba), and the like.
  • the compositions may include additional skin actives such as, but not limited to, botanicals, keratolytic agents, desquamating agents, keratinocyte proliferation enhancers, collagenase inhibitors, elastase inhibitors, depigmenting agents, anti-inflammatory agents, steroids, anti-acne agents, antioxidants, salicylic acid or salicylates, thiodipropionic acid or esters thereof, and advanced glycation end-product (AGE) inhibitors to achieve cumulative or synergistic improvements in the aesthetic appearance of the treated skin.
  • the composition may comprise at least one additional botanical, such as, for example, a botanical extract, an essential oil, or the plant itself.
  • Suitable botanicals include, without limitation, extracts from Abies pindrow, Acacia catechu, Aiisma orientate, Aloe, Amorphophallus campanulatus, Anogeissus iatifoiia, Asmunda japonica, Azadirachta indica, Butea frondosa, Butea monosperma, Cedrus deodara, Chamomile, Derris scandens, Portulaca oleracea, Eclipta prostrala, Emblica officinalis, Erythina indica, Ficus benghaiensis, Glycyrrhiza glabra, Humilus scandens, Ilex purpurea Hassk, Innula racemosa, Ixora chinensis, Ligusticum chiangxiong, Ligusticum lucidum, Mallotus philippinensis, Medemia noblis, Melicope hayesii, Mimusops ele
  • composition may comprise additional active ingredients having anti-aging benefits, as it is contemplated that synergistic improvements may be obtained with such combinations.
  • anti-aging components include, without limitation, botanicals (e.g., Butea frondosa extract); phytol, thiodipropionic acid (TDPA) and esters thereof; retinoids (e.g., all-trans retinoic acid, 9-cis retinoic acid, phytanic acid and others); hydroxy acids (including alpha-hydroxyacids and beta- hydroxyacids), salicylic acid and salicylates; exfoliating agents (e.g., glycolic acid, 3,6,9- trioxaundecanedioic acid, etc.), estrogen synthetase stimulating compounds (e.g., caffeine and derivatives); compounds capable of inhibiting 5 alpha-reductase activity (e.g., linolenic acid, linoleic acid, finasteride, and mixtures thereof); barrier function enhancing
  • retinoids include, without limitation, retinoic acid (e.g., all-trans or 13-cis) and derivatives thereof, retinol (Vitamin A) and esters thereof, such as retinol palmitate, retinol acetate and retinol propionate, and salts thereof.
  • the topical compositions of the present invention may also include one or more of the following: a skin penetration enhancer, an emollient, a skin plumper, an optical diffuser, a sunscreen, an exfoliating agent, and an antioxidant.
  • An emollient provides the functional benefits of enhancing skin smoothness and reducing the appearance of fine lines and coarse wrinkles.
  • Examples include isopropyl myristate, petrolatum, isopropyl lanolate, silicones (e.g., methicone, dimethicone), oils, mineral oils, fatty acid esters, or any mixtures thereof.
  • the emollient may be preferably present from about 0.1 wt % to about 50 wt% of the total weight of the composition.
  • a skin plumper serves as a collagen enhancer to the skin.
  • An example of a suitable, and preferred, skin plumper is palmitoyl oligopeptide.
  • Other skin plumpers are collagen and/or other glycosaminoglycan (GAG) enhancing agents.
  • GAG glycosaminoglycan
  • the skin plumper may comprise from about 0.1 wt % to about 20 wt% of the total weight of the composition.
  • An optical diffuser is a particle that changes the surface optometries of skin, resulting in a visual blurring and softening of, for example, lines and wrinkles.
  • optical diffusers examples include, but are not limited to, boron nitride, mica, nylon, polymethylmethacrylate (PMMA), polyurethane powder, sericite, silica, silicone powder, talc, Teflon, titanium dioxide, zinc oxide, or any mixtures thereof.
  • the optical diffuser may be present from about 0.01 wt % to about 20 wt% of the total weight of the composition.
  • a sunscreen for protecting the skin from damaging ultraviolet rays may also be included.
  • Preferred sunscreens are those with a broad range of UVB and UVA protection, such as octocrylene, avobenzone (Parsol 1789), octyl methoxycinnamate, octyl salicylate, oxybenzone, homosylate, benzophenone, camphor derivatives, zinc oxide, and titanium dioxide.
  • the sunscreen may comprise from about 0.01 wt % to about 70 wt % of the composition.
  • Suitable exfoliating agents include, for example, alpha-hydroxyacids, beta- hydroxyacids, oxaacids, oxadiacids, and their derivatives such as esters, anhydrides and salts thereof.
  • Suitable hydroxy acids include, for example, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid and derivatives thereof.
  • a preferred exfoliating agent is glycolic acid.
  • the exfoliating agent may comprise from about 0.1 wt % to about 80 wt % of the composition.
  • An antioxidant functions, among other things, to scavenge free radicals from skin to protect the skin from environmental aggressors.
  • antioxidants examples include compounds having phenolic hydroxy functions, such as ascorbic acid and its derivatives/esters; beta-carotene; catechins; curcumin; ferulic acid derivatives (e.g. ethyl ferulate, sodium ferulate); gallic acid derivatives (e.g., propyl gallate); lycopene; reductic acid; rosmarinic acid; tannic acid; tetrahydrocurcumin; tocopherol and its derivatives; uric acid; or any mixtures thereof.
  • phenolic hydroxy functions such as ascorbic acid and its derivatives/esters; beta-carotene; catechins; curcumin; ferulic acid derivatives (e.g. ethyl ferulate, sodium ferulate); gallic acid derivatives (e.g., propyl gallate); lycopene; reductic acid; rosmarinic acid; tannic acid; tetrahydrocurcumin; tocopherol and
  • antioxidants are those that have one or more thiol functions (-SH), in either reduced or non-reduced form, such as glutathione, lipoic acid, thioglycolic acid, and other sulfhydryl compounds.
  • the antioxidant may be inorganic, such as bisulfites, metabisulfites, sulfites, or other inorganic salts and acids containing sulfur.
  • Compositions of the present invention may comprise an antioxidant preferably from about 0.001 wt % to about 10 wt%, and more preferably from about 0.01 wt% to about 5 wt%, of the total weight of the composition.
  • vitamins such as tocopherol and ascorbic acid
  • vitamin derivatives such as ascorbyl monopalmitate
  • thickeners such as hydroxyalkyl cellulose
  • gelling agents such as bentonite, smectite, magnesium aluminum silicate and lithium magnesium silicate
  • metal chelating agents such as EDTA
  • pigments such as zinc oxide and titanium dioxide
  • colorants such as emollients; and humectants.
  • the invention provides a method for improving aging skin by topically applying a composition comprising the peptide of the invention, preferably in a cosmetically acceptable vehicle, over the affected area for a period of time sufficient to reduce, ameliorate, reverse or prevent dermatological signs of aging.
  • This method is particularly useful for treating signs of skin photoaging and intrinsic aging.
  • the improvement in the condition and/or aesthetic appearance is selected from the group consisting of: reducing dermatological signs of chronological aging, photo-aging, hormonal aging, and/or actinic aging; preventing and/or reducing the appearance of lines and/or wrinkles; reducing the noticeability of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, perpendicular wrinkles between the eyes, horizontal wrinkles above the eyes, and around the mouth, marionette lines, and particularly deep wrinkles or creases; preventing, reducing, and/or diminishing the appearance and/or depth of lines and/or wrinkles; improving the appearance of suborbital lines and/or periorbital lines; reducing the appearance of crow's feet; rejuvenating and/or revitalizing skin, particularly aging skin; reducing skin fragility; preventing and/or reversing of loss of glycosaminoglycans and/or collagen; ameliorating the effects of estrogen imbalance; preventing skin atrophy; preventing, reducing, and/or treating hyperpigmentation
  • compositions of the present invention enhance and improve the aesthetic appearance of skin by stimulation of collagen and restoring and maintaining homeostasis of this component.
  • the composition will typically be applied to the skin one, two, or three times daily for as long as is necessary to achieve desired anti-aging or therapeutic benefit.
  • the treatment regimen may comprise daily application for at least one week, at least two weeks, at least four weeks, at least eight weeks, or at least twelve weeks. Chronic treatment regimens are also contemplated.
  • trans dermal patches including single-layer, multilayer, reservoir, matrix patches and the like in the form of nose strips, peel-off paper masks, specialized treatment patches for eyes and facial contours, etc.; bandages including gauze, tube, and the like; poultices such as those used as facial masks, etc. are used to administer the cosmetic compositions of the current invention to areas of skin in need thereof for extended periods of time.
  • the peptide of the invention as active component is topically applied to an "individual in need thereof," by which is meant an individual that stands to benefit from reducing visible signs of skin damage or aging.
  • the peptide component is provided in a physiologically, cosmetically, and dermatologically-acceptable vehicle, diluent, or carrier, where the composition is topically applied to an affected area of skin and left to remain on the affected area in an amount effective for improving the condition and aesthetic appearance of skin.
  • methods for treating fine lines and wrinkles comprise topically applying the inventive peptide and compositions of the invention to the skin of an individual in need thereof, e.g., topical application directly to the fine line and/or wrinkle in an amount and for a time sufficient to reduce the severity of the fine lines and/or wrinkles or to prevent or inhibit the formation of new fine lines and/or wrinkles.
  • the effect of a composition on the formation or appearance of fine lines and wrinkles can be evaluated qualitatively, e.g., by visual inspection, or quantitatively, e.g., by microscopic or computer assisted measurements of wrinkle morphology (e.g., the number, depth, length, area, volume and/or width of wrinkles per unit area of skin).
  • This embodiment includes treatment of wrinkles on the skin of the hands, arms, legs, neck, chest, and face, including the forehead.
  • the compositions of the invention will be useful for treating thin skin by topically applying the composition to thin skin of an individual in need thereof.
  • Thin skin is intended to include skin that is thinned due to chronological aging, menopause, or photo-damage.
  • the treatment is for thin skin in men
  • other embodiments treat thin skin in women, premenopausal or post-menopausal, as it is believed that skin thins differently with age in men and women, and in particular in women at different stages of life.
  • the method of the invention may be employed prophylactically to forestall aging including in subjects that have not manifested signs of skin aging, most commonly in individuals under 25 years of age.
  • the method may also reverse or treat signs of aging once manifested as is common in subjects over 25 years of age.
  • the composition according to the invention may be in any galenical form conventionally used for topical application and especially in the form of aqueous gels, or aqueous or aqueous-alcoholic solutions.
  • a fatty or oily phase By adding a fatty or oily phase, it may also be in the form of dispersions of lotion type, emulsions of liquid or semi- liquid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous phase (O/W) or conversely (W/O), or suspensions or emulsions of soft, semisolid or solid consistency of the cream or gel type, or alternatively multiple emulsions (W/O/W or 0/W/O), microemulsions, vesicular dispersions of ionic and/or nonionic type, or wax/aqueous phase dispersions.
  • These compositions are prepared according to the usual methods.
  • the invention also relates to compositions conditioned in pressurized form in an aerosol device or in a pump-dispenser bottle; conditioned in a device equipped with a perforated wall, especially a grille; conditioned in a device equipped with a ball applicator ("roll-on"); characterized in that they contain at least perlite particles.
  • they contain the ingredients generally used in products of this type, which are well known to those skilled in the art.
  • “Physiologically or cosmetically or dermatologically acceptable medium or vehicle or carrier” is also understood to mean a medium compatible with the materials and / or human keratin fibers such as, but not limited to, skin, mucous membranes, nails, scalp and / or hair.
  • This physiologically acceptable medium comprises water, optionally in admixture or not with one or more organic solvents such as alcohols, in particular ethanol, isopropanol, tert-butanol, n- butanol, polyols such as glycerol, propylene glycol, butylene glycol and polyol ethers.
  • organic solvents such as alcohols, in particular ethanol, isopropanol, tert-butanol, n- butanol, polyols such as glycerol, propylene glycol, butylene glycol and polyol ethers.
  • composition according to the invention may also comprise a fatty phase, which may include oils, gums, waxes commonly used in the field of application considered.
  • the cosmetic compositions according to the invention may contain active ingredients, additives or excipients customary in cosmetics: biological assets (Anti-aging, fat skinning, lightening, whitening, anti-perspirant, anti-oxidant, etc.), pigments, dyes, sunscreens, polymers, film- forming, oils, fats, silicones, moisturizers, emollients, solvents, surfactants, vitamins, and / or preservatives, or other cosmetic ingredients.
  • biological assets Anti-aging, fat skinning, lightening, whitening, anti-perspirant, anti-oxidant, etc.
  • pigments dyes
  • sunscreens polymers
  • film- forming oils, fats, silicones, moisturizers, emollients, solvents, surfactants, vitamins, and / or preservatives, or other cosmetic ingredients.
  • compositions of the invention may be in the form of gels, sera, of direct emulsions, reverse or multiple sticks, poured hot, free or compacted powder, or cream.
  • composition according to the invention may be in any pharmaceutical form normally used in the cosmetic and dermatological fields.
  • aqueous, aqueous-alcoholic solution optionally gelled, a dispersion of the lotion type biphasic optionally, an oil-in-water or multiple water-in-oil emulsion, an aqueous gel, an oil dispersion (s) in an aqueous phase, in particular using spherules, these spherules to be better or polymeric particles, lipid vesicles of ionic and / or nonionic, or alternatively in the form of a powder, a serum, a paste or a flexible rod.
  • a dispersion of the lotion type biphasic optionally, an oil-in-water or multiple water-in-oil emulsion, an aqueous gel, an oil dispersion (s) in an aqueous phase, in particular using spherules, these spherules to be better or polymeric particles, lipid vesicles of ionic and / or nonionic, or alternatively
  • It can be solid consistency, pasty or liquid more or less fluid.
  • the combination is administered orally, topically or intra-dermal.
  • the composition comprises a physiologically acceptable medium for topical, oral administration or by injection, in particular intra-dermal.
  • composition is in the form of a cosmetic cream.
  • the composition is injectable and comprises an injection medium, such as one or more macromolecules of the extracellular matrix, such as hyaluronic acid, collagen, and / or cells, and / or extracted from adipose tissue or preparation containing.
  • an injection medium such as one or more macromolecules of the extracellular matrix, such as hyaluronic acid, collagen, and / or cells, and / or extracted from adipose tissue or preparation containing.
  • composition of the invention may therefore be suitable for injection intra-skin by use of a syringe, using one or more micro-needles, or an implant or a biodegradable reservoir.
  • bio-degradable container indicates a device comprising degradable molecules in a more or less long time, often a few weeks to several months or even years after introduction into the skin.
  • compositions can be used the active ingredients of the invention in combination with a extra-cellular medium injection such that one or more of the matrix macromolecules such as hyaluronic acid or collagen, or cells adipose tissue extracts or preparations containing it.
  • matrix macromolecules such as hyaluronic acid or collagen, or cells adipose tissue extracts or preparations containing it.
  • extracts of adipose tissue from the body of the subject receiving the injection of the composition of the invention may be otherwise.
  • This extract can be from another subject or group of subjects.
  • the invention further relates to an injection device prefilled with the combination or composition according to the invention intra-cutaneous.
  • the invention also relates to an intra-dermal injection kit comprising an intra-dermal injection device, such as a syringe, using one or more micro-needles, an implant or a biodegradable container, said kit comprising also the composition physically separated according to the particular injection device for filling injection just before the injection device invention.
  • an intra-dermal injection device such as a syringe
  • micro-needles such as a syringe
  • implant or a biodegradable container said kit comprising also the composition physically separated according to the particular injection device for filling injection just before the injection device invention.
  • the invention also relates to the cosmetic use (for aesthetic purposes) of the peptides of the invention for preventing and / or treating the signs of skin aging and / or photo-aging, in particular to reduce ptosis, reduce the loss of skin firmness, reduce visibility of wrinkles or skin wrinkles, or improve the quality and / or the transparency of the skin.
  • the use is exclusively aesthetic treatment of a subject, preferably human, and therefore excludes any surgical treatment, or therapy.
  • the invention is ultimately decreasing the visibility of wrinkles, thus justifying the use of this aesthetic goal.
  • Means reduce the visibility of wrinkles reducing the visibility or the depth of the grooves formed at the wrinkles of the skin surface, visually assessed by a human or by means of a suitable apparatus to quantify the appearance of skin wrinkles.
  • the present invention also provides a method of cosmetic care for immediate filling wrinkles, characterized in that it comprises the topical application, oral administration or intra-dermal injection of a composition according to the invention.
  • This topical or intra-dermal injection is performed at the zone to be filled, or nearby.
  • this application or injection at least 2 cm, and preferably less than 1 cm, parallel to the filling lines is performed.
  • the composition of the invention is an emulsified cosmetic composition comprising a water and an oil phase, and dispersed in the water an oil phases (a) at least one peptide of general formula (I), (b) optionally an additional anti-aging agent retarding the effects of natural aging and free radical activity on the skin's complexion, and (c) a sunscreen retarding the effects of aging due to sunlight on the skin's complexion.
  • the emulsified cosmetic composition further comprises (d) a preservative for preventing microbial growth in the composition, (e) a thickener to increase the viscosity of the composition, (f) an antioxidant to prevent rancidity and discoloration of the composition, and (g) an emulsifier for combining the water phase and oil phase ingredients as well as for giving the composition a suitable texture when applied to the skin.
  • the anti-aging agent is selected from the group consisting of proteins, flavonoid compounds, glycogen, and combination thereof.
  • the proteins can comprise serum proteins and hydrolyzed animal proteins.
  • the flavonoid compounds can be provided by extracts of the plants butcher broom, buckwheat, and passion flower.
  • the sunscreen can be selected from the group consisting of paraminobenzoic acid derivatives, oxyphenones, benzophenones, titanium dioxide, zinc oxide, cinnamate, and combinations thereof.
  • the preservatives can be propylene glycol, trisodium EDTA, Phenonip , a preservation complex, and combinations thereof.
  • the preservation complex contains methylparaben, propylparaben, 2-bromo-2-nitropropane-l,3diol, and hexamidine diisethionate.
  • Phenonip is a commercially available preservative and contains phenoxyethanol, methylparaben, ethylparaben, propylparaben, and butylparaben.
  • the antioxidant can be Tenox II and/or ascorbyl palmitate. Tenox II is a commercial product which contains propylene glycol, butylated hydroxyanisole, propyl gallate, and citric acid.
  • Preferred thickeners are xanthan gum and carrageenan.
  • the composition can also contain one or more colorants, such as titanium dioxide. Anti-aging ingredients
  • the agents for retarding the effects of natural aging and aging due to free radical activity on the skin's complexion are selected from the group consisting of proteins, flavonoid compounds, glycogen, and combinations thereof.
  • the emulsified composition of the present invention employs a commercial source for the proteins, flavonoid compounds, and glycogen. These ingredients are supplied in various combinations in two products, called Flavoplasmine LS2704 P2 (hereinafter Flavoplasmine) and Iconoderm LS 1054 B (hereinafter Iconoderm), both available from Lab Serobiiquess, Inc., Somerville, New Jersey.
  • Flavoplasmine The active ingredients of Flavoplasmine are serum proteins, hydro lyzed animal proteins and flavonoid compounds.
  • the flavonoid compounds within the Flavoplasmine are furnished by butcher broom extract, buckwheat extract, and passion flower extract.
  • the Flavoplasmine further contains a mono stearatepoly ethylene glycol ester of stearic acid (PEG 6-32) serving as an emulsifier, and glycerin functioning as a solvent.
  • PEG 6-32 mono stearatepoly ethylene glycol ester of stearic acid
  • Iconoderm comprises serum proteins, hydrolyzed animal proteins, and glycogen. Sodium lactate is present for buffering. Iconoderm further contains sodium pyrrolidone carboxylate.
  • the anti-free radical ingredients of the emulsified cosmetic composition can be selected from the group consisting of derivatives of vitamins C or E, selenium metal compounds, or beta carotene derivatives. Sunscreens
  • the agents protecting against aging of the skin's complexion due to sunlight are one or more of the sunscreens identified in the following table.
  • a satisfactory cosmetic composition comprises octyldimethyl paraminobenzoic acid and benzophenone-3. Preservatives
  • the emulsified cosmetic composition can be prepared with propylene glycol, trisodium EDTA, a commercially obtained composition known as Phenonip , and a preservation complex.
  • the preservation complex is a white, colorless, crystalline powder. It contains methylparaben, propylparaben, 2-Bromo-2-Nitropropane-l ,3-Diol, and hexamidine isethionate.
  • Phenonip is a practically colorless viscous liquid mixture of phenoxyethanol, methylparaben, ethylparaben, propylparaben, and butylparaben available from Nipa Laboratories, Inc., Wilmington, Del.
  • an antioxidant may be included in the cosmetic composition.
  • Tenox II is obtained from Eastman Chemical Products, Inc., Kingsport, Tenn. and contains propylene glycol, butylated hydroxyanisole, propyl gallate, and citric acid.
  • Emulsifiers serve two functions in the present invention. They act like an emulsifier to combine the water soluble and non-water soluble phases together; that is, form a stable bridge between the waters and the oils of the ingredients.
  • the emulsifiers also serve as emollients, providing a pleasant, aesthetically appropriate, tactile feeling when the emulsified composition is applied to the skin.
  • the preferred cosmetic composition comprises the preferred emulsifiers namely isodecyl oleate, CI 2- 15 alcohols benzoate, arachidyl propionate, octyldodecyl stearoyl stearate, steareth-2, PEG-8 stearate, isoceteth-20, steareth-21 , acetylated lanolin, dimethicone and 3-cyclohexene-l- methanol .alpha., 4-dimethyl-.alpha.(4-methyl-3-pentenyl) (hereinafter "bisabolol").
  • bisabolol 4-dimethyl-.alpha.(4-methyl-3-pentenyl)
  • the composition contains sufficient thickener that the cosmetic composition does not run off the face and other skin areas when applied.
  • the thickeners complement the function of the emulsifiers in holding together the water and oil phases of the composition.
  • the emulsified composition preferably employs as thickeners xanthan gum and carrageenan.
  • Demineralized water is preferably used in the cosmetic composition as the emulsified base.
  • the cosmetic composition can include at least one colorant, preferably titanium dioxide.
  • Peptides of general formula (I) were prepared by Fmoc/tBu methodologies using a polystyrene resin (PS) or PEG-PS resin derivatized with Rink amide for the production of carboxyamide peptides at the C-terminus.
  • the synthetic strategy is based on the elongation of the peptide starting from the C-terminus, through sequences of deprotection of the a-amino group and acylation with the activated carboxylic group of the subsequent amino acid.
  • acid labile protecting groups such as Boc for lysine (Lys(Boc)), ornitine (Orn(B
  • the acetyl, palmitoyl or other acyl groups were introduced by conventional chemistry, for example by reaction with anhydrides or by coupling in the presence of HO At and DIPC in DMF. The completion of the reaction was followed by using the Kaiser test in the presence of ninhydrin to evaluate the presence of free amino groups.
  • the peptides precipitated from cold tert-butyl-methyl ether and were centrifuged at 3600rpm for 30 min at 4°C. The precipitated peptide was washed 3 times and finally freeze dried with a mixture of H 2 0/CH 3 CN containing 0.1% trifluoroacetic acid.
  • All peptides were purified by reverse phase high pressure liquid chromatography (RP-HPLC) using the WATERS 2489 chromatographic pump or alternatively the WATERS 600/E coupled with a detector WATERS 2545 or alternatively a JASCO UV-975 detector, using chromatographic columns X-Bridge BEH130 Perp C18 ⁇ (19x150 mm) or X-Bridge BEH130 Perp C18 5 ⁇ (30x150 mm). All the peptides were characterized before and after purification by using a Waters UPLC/MS ACQUITY system equipped with mass spectrometer Waters SQ. All peptides were obtained, after liophilization, with purity > 95%.
  • RP-HPLC reverse phase high pressure liquid chromatography
  • the exchange of the trifiuoroacetate counterion depends on the selected counter ion (acetate, chloride, etc).
  • the preparation of peptides with acetate as counter ion was done through ionic exchange on resin ToyoPeral DEAE 650C, by conditioning the resin with acetic acid and eluting the peptides preloaded in water with a 40 fold excess of resin/DEAE with respect to the peptide equivalents.
  • the peptides as eluted are directly freeze dried.
  • the preparation of peptides as chlorohydrates was carried out by solubilizing the freeze dried peptides with a 1 mM solution of HC1 followed by liophilization. This operation can be repeated more than ones: completion of the exchange is followed by F 19 -NMR analysis.
  • Liophilized peptides are stored at -20°C.
  • Negative controls are carried out under the same conditions but in the absence of the peptide.
  • the signals of the immunostained collagen type I were obtained by laser excitation at 488 nM and an excitation filter at 540 nM. Only object with dimensions greater than average were considered as cells. The sum of signals from single objects for each sample was normalized to the nucleus count described before. The so obtained value represents, in relative fluorescence units per cells, the induction of collagen type I due to treatment.
  • Fluorescence microscopy was used to quantify and localize within the cell the induced collagen type I. Toward this end the instrument InCell 1000 (GE Healthcare, USA) was used. Fluoro fores excitation, both nuclear and from immunostained collagen, was done with a Xenon lamp. The incoming and outgoing light was filtered by using a set of filters similar to that used for the cytometer. For each sample ten images per two channels were acquired via CCD camera: one originated from the excitation of the nuclear marker and one originated from the excitation of the fluorophores used for the immuno staining of collagen type I. The quantification of the latter was done by image analysis (lOx phase objective) with the InCell Investigator software (GE Healthcare, USA). The signal related to collagen type I is localized within cytoplasmic vesicles, probably due to the fact that this protein is secreted. The analysis protocol is as follows:
  • the average of signals for each sample represents the induction level of collagen type I per cell which is triggered by the treatment.

Abstract

The present invention refers to peptides of general formula (I) and their salts for dermatological and cosmetic use, in particular for preventing or reducing the signs of cutaneous aging and for the treatment of wounds (i.e. burns, lacerations), skin ulcers, chronic wounds or bedsores.

Description

DERMATOLOGICAL AND/OR COSMETIC PEPTIDES FOR USE IN SKIN
TREATMENT
FIELD OF THE INVENTION
The present invention relates to peptides capable of increasing the amount and/or the stability of collagen or of collagen-like molecules in eukaryote cells, in particular cells of the derm and/or epiderm, more particularly human fibroblasts. The peptides of the invention can be used in a variety of dermatological applications, such as the treatment of burns, wounds, lacerations, skin ulcers, chronic wounds or bedsores. Further, peptides of the invention can be used for cosmetic treatment to improve the general state of the skin and to prevent and/or reduce signs of skin aging.
BACKGROUND
Skin represents the covering, or integument of the body's surface that provides protection and receives sensory stimuli from the external environment. It consists of a series of tissues of ectodermal and mesodermal origin and can have various colors, physiological and organic structure, subjected to more or less visible aging processes. As a mediator between the body and the outside world, the skin performs several functions in vertebrates. In particular, the skin has a protective function, being an anatomical barrier, it is the first line of defense against external aggression. In addition, the skin has a key role in sensitivity, through the presence of thermoreceptors, pressoreceptors and algoceptors, in thermal regulation and in controlling evaporation. The skin consists of three layers of tissue: the epidermis, an outermost layer that contains the primary protective structure, the stratus corneum; the dermis, a fibrous layer of connective tissue that supports and strengthens the epidermis; and the subcutis, a subcutaneous layer of fat beneath the dermis that supplies nutrients to the other two layers and that cushions and insulate the body. The skin also contains a number of elements that complement its structure and function: hair, nails and glands of various kinds.
Superficial epidermis is a stratified epithelium largely composed of keratinocytes that are formed by division of cells in the basal layer, and give rise to several distinguishable layers as they move outwards and progressively differentiate. Within the epidermis, there are several other cell populations, namely melanocytes, which donate pigment to the keratinocytes, Langerhans' cells, which have immunological functions and Merkel cells. The epidermis can be divided into four distinct layers: stratum basale or stratum germinativum, stratum spinosum, stratum granulosum and stratum comeum. Keratinocytes are the most abundant cell type in the epidermis.
The dermis is the connective tissue component of the skin, consisting of cells and extracellular matrix or ground substance. Cell types include fixed cells, such as fibroblasts, mast cells, macrophages or histiocytes and fat cells, and the mobile cells originating from blood. Fibroblasts are the most abundant and important cell type of the dermis, as they produce fibrous structures - collagen - and the macro molecular components of the ground substance.
The basis of the dermis is a supporting matrix or ground substance in which polysaccharides and protein are linked to produce macromolecules with a remarkable capacity for retaining water. Within and associated with this matrix are two kinds of proteins: collagen, which has great tensile strength and forms the major constituent of the dermis, and elastin, which makes up only a small proportion of the bulk. The cellular constituents of the dermis include fibroblasts, mast cells and histiocytes (monocyte/macrophages). The dermis has a very rich blood supply, although no vessels pass through the dermal-epidermal junction
The extracellular matrix consists of fibers and amorphous component. The fibers form the support structure of the dermis and ensure dermis tension/traction resistance and flexibility. The extracellular matrix is the largest component of normal skin and gives the skin its unique properties of elasticity, tensile strength and compressibility. Collagen is the major insoluble fibrous protein in the extracellular matrix and in connective tissue, accounting for about 75% of the weight of dry dermis. From a biochemical point of view, fibers are composed of collagen, a fibrous protein whose synthesis begins in fibroblasts and is completed in the extracellular matrix. The collagen fibrous component is poorly extensible; however, it is extremely tough and especially resistant to tension parallel to the fibers. This characteristic is important in maintaining the dynamic strength of the skin.
Collagen fiber molecules are produced in the rough endoplasmic reticulum of fibroblasts. Helical procollagens with three alpha-c ai are secreted and the molecular ends are cut by procollagen peptidase to become tropocollagens. The molecules are crosslinked to each other with a regular gap that forms the striped collagen fiber. The collagen molecule is formed by said tropocollagen simple protein units, organized in three amino acid chains, forming a triple spiral shaped rope: this confers rigidity and strength to the fibers, thus responsible for the function of supporting and holding the dermis. In the process of formation of collagen fibers, the elementary units are associated with each other in the intercellular spaces to form filamentous structures even more complex named microfibrils and fibrils, which in turn are organized in a three-dimensional network and take the name of reticular fibers. By assembly of several bundles of fibrils, are formed the real collagen fibers, which run parallel to the skin surface, intersecting each other at right angles.
Collagen is a family of inert proteins but characterized by a continuous turnover. The fibrous organization of dermal proteins and the type of molecular interactions determine the biomechanical properties (elasticity, resistance) of the skin. Nonspecific proteases transform the collagen polymer in monomers, whose degradation is due to a specific collagenase. Proteolytic degradation of collagens by proteases produces small fragments which are released systemically. Degradation products of collagen are further cleaved by specific proteases, namely cathepsins, and subsequently phagocytosed by macrophages. Chronologically, aged skin shows decreased production of new collagen and increased proteolytic activity resulting in increased collagen degradation. Senescent fibroblasts show decreased synthesis of type I collagen and proliferate at a much slower rate than fibroblasts in young skin.
With age, collagen undergoes modifications both qualitative (structural and physical properties, i.e the fibers become less extensible and insoluble) and quantitative (decrease of amount and increase of the size or thickening of each fiber), resulting in a reduction of skin elasticity. The loss of elasticity of the skin is the main responsible for the formation of wrinkles. Skin aging is therefore a process of degradation of the cutaneous tissue that requires a real reconstruction.
Skin aging is a process influenced by extrinsic and intrinsic factors and is manifested by a progressive loss of skin tissue, gradual loss of skin elasticity and the appearance of fine lines and wrinkles. Intrinsic factors include the decrease in cutaneous blood flow, a loss of cells and cell function, the continuous generation of reactive oxygen species (ROS) during cellular metabolism and genetic predispositions. Extrinsic factors include exposure to UV-light, environmental pollution, cigarette smoke and extreme weather conditions.
In a similar way, the cicatrization (or healing) is the process of reconstruction of the cutaneous tissue, damaged due to an injury of any nature.
Structural reconstruction of dermal elastic fibers is an important action against skin aging. Moreover, the aging and wound healing are linked processes as they both share common molecular targets.
Wound healing has three principal phases: inflammatory, proliferative, and remodeling. The inflammatory phase begins at the time of injury. Next, fibroblasts proliferate to become the dominant cell of the proliferative phase. They produce collagen, which provides structure to the wound and replaces the fibronectin-fibrin matrix. Angiogenesis of new capillaries occurs to sustain the fibroblast proliferation. Keratinocytes also epithelialize the wound. The remodeling phase begins at about 2 to 3 weeks and can last up to 2 years. At this time, collagen synthesis and degradation reach equilibrium. The correction of skin aging and dermal cicatrization -share the same sites of action in the dermis. The cicatrization is then the result of the repair of a skin tissue. The signs of aging - can be considered as long-term skin lesions that require a real tissue reconstruction, through a process of cicatrization able to act on the mechanisms of collagen synthesis and on their organization. It is known that natural peptides play an important role in coordinating the biochemical processes and are able to stimulate the synthesis of collagen. For this reason, many peptides are important ingredients for the preparation of agents that can improve the general state of the skin.
Research on wound healing produced much of the evidence showing the importance of peptides in improving the signs of aging. The design of peptides that either stimulate collagen production or down regulate collagenase activity could cosmetically benefit aging skin. These peptides are small- sequence amino-acid chains that may stimulate angiogenesis, production of granulation tissue, and new collagen synthesis. Three classes of peptides have been designed with these goals in mind: a) signal peptides, triggering wound healing mechanisms that activate fibroblasts; b) carrier peptides, capable of delivering copper into skin resulting in activation of enzymatic wound healing pathways; c) neurotransmitter-inhibiting peptides, interfering with stabilization step in neurotransmitter release.
With the term matrikines are indicated peptides obtained by (partial) proteolytic cleavage of constituents of the extracellular matrix that are able to regulate positively or negatively cell activities and thus have activities similar to cytokines or regulatory peptide factors. This class of mediators plays a significant role in physiological or pathological processes (like angiogenesis, wound healing, tumor invasion) by modulating, for example, chemotaxis, mitogenesis, and responses to stimuli causing cell death. Matrikines are important components of a broad cell and tissue sensory system to detect and respond to various types of tissue injury (Maquart FX et al Biochimie. 2005; 87, 3-4, 353-60).
It is known that the synthesis of collagen can be stimulated in a dose dependent manner by the C- terminal fragment of Collagen type I, having sequence Lys-Thr-Thr-Lys-Ser, (Katayama K. et al., J. Biol. Chem. 1993, 259, 9941-9944). WO2000/015188 relates to the use of peptides of formula X-Thr-Thr-Lys-Y in cosmetic or dermopharmaceutical compositions. Such peptides are chemically modified to increase their lipophilicity by grafting on the N-terminal amine of X either a fatty acid chain or by esterification or amidation of the C-terminal carboxyl group of Y.
WO91/12014 describes the biological activity of the copper complex of the tripeptide Gly-His-Lys (GHK-Cu). This peptide increases fibroblastic collagen synthesis thereby enabling a more rapid replacement of the transplants with human tissue. It is present in plasma, saliva and urine in physiologically active amount and it has two main functions: (1) first as a potent tissue protective, anti-inflammatory agent that limits oxidative damage after tissue injury, and (2) as a signal that activates tissue remodeling, consisting of removal of damaged protein and scar tissue and replacement by normal tissue. Thus, GHK-Cu links the processes of removal of damaged scar tissue and deposition of new tissue (Methods in Enzymology, Vol 147, 1987, pp 314-328, Academic Press).
WOOl/43701 relates to the use of N-palmytoyl-Gly-His-Lys (Palm-GHK) tripeptide in cosmetic or dermopharmaceutical composition for preventing or reducing the signs of skin aging.
W099/12968 and WO00/42071 disclose tripeptides of formula KXK, with X one of the twenty naturally occurring amino acids, and their use in therapeutic methods to inhibit or augment inflammatory response. WO 2010/082177 proposes other peptidic compounds of formula KXK for the cosmetic and dermopharmaceutic fields, that are able to improve the general state of the skin and its appendages.
Peptides for use in the treatment and prophylaxis of inflammation of epithelial tissues and their use also for non therapeutic skin care are described in WO 2012/107244.
WO00/43417 discloses the use of certain peptides and derivatives as cosmetics or pharmaceutical compositions for the regulation of impaired immunologic functions and inflammations of the skin. Exemplified peptides include N-Palm-Gly-Gln-Pro-Arg (Palm-GQPR). The combination of Palm- GQPR and Palm-GHK is produced by Sederma under the trademark Matrixyl™ 3000. The synergistic effect of the two peptides is described in WO2005/048968.
Further peptides capable of chelating copper and complexes copper-peptide useful in promoting healing and in improving skin appearance are described for example in EP0189182; EP0190736; US 4,877,770; EP0314755; EP0288278. Some copper-peptide complexes demonstrated efficacy in promoting hair growth. Some examples are described in US 5,177,061 ; US 5,120,831; US 5,214,032; US 5,538,945; US 6,017,888.
Human angiogenin (hAng) is a 14 KDa single polypeptide chain that belongs to the pancreatic ribonuclease superfamily which is normally present in the plasma. This protein promotes the formation of new blood vessels from pre-existing blood vessels through a process called angiogenesis. Phyisiologically, angiogenin is induced during inflammation, exhibiting wound healing properties as well as microbiocidal activity and conferring host immunity (Tello-Montoliu A et al, J. Thromb. Haemost. 2006, 4, 1864-1874).
EP622071 describes a whitening cosmetic preparation containing angiogenin for preventing the developments of blotches and freckles of skin through the inhibition of melanin production.
JP2004331566 describes a skin collagen production enhancer comprising angiogenin and/or an angiogenin degradate obtained by decomposing angiogenin with a protease such as pepsin or pancreatin. It is further described a drink preparation for enhancing collagen production, comprising angiogenin obtained from skim milk, useful for preventing skin roughness, wrinkles, decreased elasticity, etc.
La Mendola et al. (Dalton Trans, 2010, 39, 10678-10684) found that the activity of angiogenin may be affected by the presence of copper ions and that angiogenin is able to bind tightly 2.4 copper ions per molecule, the copper ions increasing the affinity of angiogenin to endothelial cells and modulating its interaction towards endothelial cells. Copper(II) complexes with the peptides encompassing the putative endothelial cell binding domain of angiogenin, Ac-KNGNPHREN-NH2 and Ac-PHREN-NH2, have been characterized by potentiometric, UV-vis, CD and EPR spectroscopy methods. The coordination features of all the copper complex species derived by both peptides are practically the same, as predictable because of the presence of a proline residue within their aminoacid sequence. In particular, La Mendola et al found that Ac-PHREN-NH2 is the minimal real aminoacid sequence involved in the binding of copper(II).
However, there is the still for peptides that have benefits for the skin, particularly advantageous peptides can increase the amount or stability of collagen.
Within the present invention, it has been unexpectedly found that peptide Ac-PHREN-NH2 and structurally related peptides comprising from four to six aminoacids are effective in increasing amount and/or stability of collagen in the skin. In particular, peptides of the invention promotes the production of collagen type I in human fibroblasts cell cultures, and are not toxic for the cells when tested at high concentration. Such compounds are therefore particularly useful in the cosmetic and dermopharmaceutic fields, to improve the general state of the skin and to treat conditions ameliorated by an increased production and/or stability of collagen. Therefore, compounds of the invention can be used to prepare topical dermopharmaceutical and/or cosmetic compositions useful for facilitating wound healing, treatment of minor burns, and to prevent signs of cutaneous aging and photoaging.
DESCRIPTION OF THE INVENTION
In the present invention it was unexpectedly found that a novel class of peptides is capable of increasing in a substantial significant way the synthesis of collagen type I in human fibroblasts cell cultures. At the effective concentrations, the peptides of the invention do not show cellular toxicity. The newly formed collagen fibers are very important for accelerating the process of cutaneous regeneration.
It is an object of the present invention a peptide of general formula (I):
Figure imgf000008_0001
m, n, p, q = 0, 1 and if one of m, n, p o q is 0, the others are 1 ;
y = 0, l, 2; z = 0, 1;
Ri = H, OH;
A = H, R2CO wherein R2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
X is selected from an amino acid, an amino acid derivative, OR3, NR3R4 wherein R3 and R4 are each independently selected from H, C1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof, with the exclusion of the compounds wherein:
Ri = H; m = n = p = q = l ; y = l; z = 0; X = NH2.
Preferably A is selected from acetyl and palmitoyl.
Preferably the peptide of general formula (I) is selected in the group consisting of:
- Acetyl-Pro-His-Arg-Glu-Asn-Lys-(N8-Palmitoyl) (SEQ ID No. 1); - Acetyl-Pro-His-Arg-Glu-Gln (SEQ ID No. 2);
- Acetyl-Pro-His-Arg-Glu;
- His-Arg-Glu-Asn;
- Acetyl-Pro-Arg-Glu-Asn;
- Acetyl-Hyp-His-Arg-Glu-Asn (SEQ ID No. 3);
- Acetyl-Pro-His-Glu-Asn.
It is a further object of the present invention the peptide of general formula (I),
Figure imgf000009_0001
wherein:
m, n, p, q = 0, 1 e and if one of m, n, p o q is 0, the others are 1 ;
y = 0, l , 2; z = 0, 1;
Ri = H, OH;
A = H, R2CO wherein R2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
X is selected from a amino acid, an amino acid derivative, OR3, NR3R4 wherein R3 and R4 are each independently selected from H, C1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof,
for medical use. Preferably for use in the treatment of skin conditions which are ameliorated by an increase of the amount of collagen, still preferably for use in promoting wound healing and in the treatment of burns, skin ulcers, chronic wounds, bedsores.
In a preferred embodiment in the peptides for use as indicated above, A is selected from acetyl and palmitoyl. The peptides of the invention are also for use in the treatment of skin conditions which are ameliorated by an increase of the stability of collagen. The peptides of the invention are also for use in the treatment of skin conditions associated with loss of collagen.
The term "treatment" comprises both a therapeutic and a preventive affect.
In a preferred embodiment, the peptide of general formula (I) for use as indicated above is selected in the group consisting of:
- Acetyl-Pro-His-Arg-Glu-Asn-Lys-(N8-Palmitoyl);
- Acetyl-Pro-His-Arg-Glu-Gln;
- Acetyl-Pro-His-Arg-Glu;
- His-Arg-Glu-Asn;
- Acetyl-Pro-Arg-Glu-Asn;
- Acetyl-Hyp-His-Arg-Glu-Asn;
- Acetyl-Pro-His-Glu-Asn;
- Acetyl-Pro-His-Arg-Glu-Asn (SEQ ID No. 4);
- Palm-Pro-His-Arg-Glu-Asn.
It is an object of the invention the use of a peptide of general formula (I) to prevent and/or reduce the cutaneous signs of aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress.
It is a further object of the invention a composition comprising at least one peptide of general formula (I) and a dermatologically acceptable vehicle.
Preferably the peptide of general formula (I) is present in said composition at a concentration between 0.0005% w/w and 0.5% w/w. In a further preferred embodiment said composition comprises at least one additional ingredient selected from the group comprising vitamin C, derivatives of vitamin C, vitamin E, derivatives of vitamin E, tocopherols, vitamin A, derivatives of vitamin A, retinal, retinoic acid.
Preferably, the composition further contains at least one other active ingredient. Still preferably, the at least one other active ingredient is selected from the group consisting of: an anti-radical agent or antioxidant.
Preferably the composition is in the form of a cream, emulsion, dispersion, gel, ointment, lotion. Preferably said composition is for use in the treatment of skin conditions which are ameliorated by an increase of the amount of collagen. It is a further object of the invention the use of said composition to prevent and/or reduce the cutaneous signs of aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress.
It is a further object of the invention a method for the treatment and/or prevention of a skin condition that is ameliorated by an increase of the amount and/or stability of collagen comprising at least one step that consists in applying, to the skin or scalp, at least one peptide as defined above or at least one composition as defined above.
It is a further object of the invention a method for the treatment and/or prevention of the skin in order to combat the signs of skin aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress, comprising at least one step that consists in applying, to the skin or scalp, at least one peptide as defined above or at least one composition as defined above.
Within the meaning of the present invention, the terms "peptide" or "polypeptide" are not particularly restricted, and in generale designate natural or synthetic peptides containing only natural amino acids, only non-natural amino acids, or combinations of natural and non-natural amino acids. In the context of the present invention, the term "peptide" denotes a chain of amino acids linked together via a peptide bond (or amide bond).The term "amino acid" as employed herein includes and encompasses all of the naturally occurring amino acids, either in the D-, L-, alio, or other stereoisomeric configurations if optically active, as well as any known or conceivable non-natural, synthetic and modified amino acid.
The term "natural amino acids" denotes the following 20 amino acids in their laevorotatory (L) or dextrorotatory (D) form, preferably in their natural L form:
Name One-letter code Three-letter code
Alanine A Ala
Arginine R Arg
Asparagine N Asn
Aspartate D Asp
Cysteine C Cys
Glutamate E Glu
Glutamine Q Gin
Glycine G Gly
Histidine H His
isoleucine 1 lie
Leucine L Leu
Lysine K Lys
Methionine M Met
Phenylalanine F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Tryptophan W Trp
Tyrosine Y Tyr
Valine V Vai
The term ' ydrophobic amino acid" denotes one of the following amino acids: I, L, V, M, F, Y, W, T, G, C or A. The term "alkaline hydrophilic amino acid" denotes one of the following amino acids: R, K or H. The term "neutral hydrophilic amino acid" denotes one of the following amino acids: S,P, N or Q. The term "acidic amino acid" denotes one of the following amino acids: D or E. In particular, the term "polypeptides" as employed herein includes and encompasses oligopeptides, peptides, polypeptides and derivatives thereof, peptide analogs and derivatives thereof, as well as pharmaceutically acceptable salts of said compounds. The term "peptides" as employed herein includes complexes with other species, such as metal ions (like copper, zinc, manganese, magnesium etc.). According to preferred embodiments of the invention, particularly interesting are the complexes with copper ions as the peptide of sequence AC-PHREN-NH2 represents the aminoacidic sequence involved in the binding of angiogenin to copper(II).
The terms "hexapeptide", "pentapeptide", "tetrapeptide" indicate compounds including a sequence of, respectively, six, five and four amino acids in consecutive order. These amino acids are indicated using the three or one letter codes, according to international conventions, from the N- terminus to the C-terminus. According to said conventions, proline is indicated as Pro or P, histidine is indicated as His or H, arginine as Arg or R, glutamic acis as Glu or E, asparagine as Asn or N, lysine as Lys or K, glutamine as Gin or Q, aspartic acid as Asp or D. The abbreviations used for the amino acids follow the rules of the Commission on Biochemical Nomenclature IUPAC-IUB specified in Eur. J. Biochem. (1984) 138, 9-37 and in J. Biol. Chem. (1989) 264, 633-673.
According to specific embodiments of the invention, the non natural amino acids include, without limitations, the hydroxyproline (Hyp), the L-l,2,3,4-tetrahydroisoquinolin-3-carboxylic acid (Tic), azetidine, D-proline (pro), homo-proline (hPro), thienylalanine (Tlia), tiazolidinalanine (Thz), ornitine (Orn), nor-arginine (Agb).
Peptides of the invention includes, without limitations: Ac-Pro-His-Arg-Glu-Asn, Ac-Pro-His-Arg- Glu-Asn-Lys-NEPalm, Palm-Pro-His-Arg-Glu-Asn, Ac-Pro-His-Arg-Glu-Gln, Ac-Pro-His-Arg- Glu, His-Arg-Glu-Asn, Ac-Pro-Arg-Glu-Asn, Ac-Hyp-His-Arg-Glu-Asn, Ac-Pro-His-Glu-Asn.
To increase the bioavailability and the capacity of the peptides of the invention to cross the skin barriers, their lipophilicity or lipophilic character can be increased through acylation of the N- terminal amino group of the peptide, or through esterification of the carboxy terminal with an alcohol, linear or branched, saturated or unsaturated, hydroxylated or not, or through both said chemical modifications.
In a preferred embodiment, N-acyl groups are acetyl, lauroyl, miristoyl, palmitoyl, steroyl, oleoyl, lineoyl. Particularly preferred are the groups N-acetyl and N-palmitoyl.
When used at the N-terminal of a sequence, "Ac" indicates an N-acyl derivative (indicated also as acyl-derivative). Similarly, "Palm" indicates a N-Palmitoyl derivative. When used at the C- terminus of a sequence, "OAlk" indicates an ester group attached to the C-terminus carboxylic group.
The polypeptides of the invention can be obtained from chemical or enzymatic synthesis starting from the constitutive amino acids or from their derivatives; alternatively, they can be obtained from natural proteins by hydrolysis under mild conditions, or by biotechnology. For example, known methods of peptide synthesis can be applied, as the Fmoc/tBu method in solid phase. Other chemical methods include Boc/bzl or liquid phase synthesis. References for the synthetic methodologies are described for example in: Solid Phase Peptide Synthesis (1984), Pierce Chemical Company, Rockford, Illinois; The Practice of Peptide Synthesis (1984), Springer Verlach, New York; Chemical Approaches to the Synthesis of Peptides and Proteins (1997), CRC, Boca Raton, FL; J. Biol. Chem. (1980), 255, 8234-8238.
The polypeptides of the invention can form homogeneous or mixed salts with mono- or polivalent acids, preferably with inorganic acids or with appropriate aliphatic carboxylic acids saturated or unsaturated, or with aromatic carboxylic acids, or with aliphatic or aromatic solfonic acids, preferably acetic acid, lactic acid and/or chloridric acid.
The compounds of the invention stimulate metabolic functions of skin cells. The compounds of the invention increase the amounts of the proteins constituent of the extracellular matrix, probably by increasing their synthesis. In particular, compounds of general formula (I) are capable of increasing the amount of collagen type (I) in human fibroblast cell cultures and have a positive effect on tissue regeneration.
The peptides of the invention find use as active ingredients for the preparation of compositions preferably for dermatologic and/or cosmetic use. Said compositions can be used for example to prevent or reduce signs of skin aging, in particular wrinkles, and/or to ameliorate skin appearance. It has been discovered that the use of at least one peptide in accordance with the present invention in cosmetic, personal care or dermopharmaceutical compositions has anti-aging activity. Anti- aging activity means some degree or capacity for treating, preventing or ameliorating one or more signs, symptoms, and/or causes of skin aging. The present peptides retard the deleterious effects of natural aging, free radical activity, and sunlight on the complexion of the skin. Another embodiment of the invention consists of the use of the peptides or of a composition comprising at least one peptide to prevent or treat the cutaneous signs of aging. The "cutaneous signs of aging" include, but are not limited to, any visible manifestations on the skin caused by aging. In particular, this means wrinkles, fine lines, chapped skin, enlarged pores, imperfections, losses in firmness, discoloration, aged areas, keratosis, losses in collagen, and other changes to the dermis and epidermis, etc. "Cutaneous signs of aging" also means any changes to the outer appearance of the skin and skin appendages caused by aging, such as superficial roughness of the corneal layer, fine lines and wrinkles, but also any internal change to the skin which is not translated systematically into a modified outer appearance, such as thinning of the dermis or any other internal degradation of the skin following exposure to ultraviolet (UV) radiation. More specifically, the invention relates to the use of the composition as described above in order to reduce the signs of skin fatigue.
The use as anti-aging is for preventing and/or treating aged or senescent skins, and/or for preventing and/or treating wrinkles and/or fine lines and/or cracks, thinning of the skin, in particular of the dermis, and/or senescence spots, and/or for combating skin disorders induced by an oxidative stress chosen from a dull appearance of the complexion, and/or hyperpigmentation of the skin, and/or a loss of quality of the sebum, and/or a dandruff appearance of the scalp, and/or a feeling of discomfort on the scalp.
The present invention provides peptides and compositions for topical application which comprise an effective amount of a peptide of the invention to treat, reverse, ameliorate and/or prevent signs of skin aging. Such signs of skin aging include without limitation, the following:
(a) treatment, reduction, and/or prevention of fine lines or wrinkles,
(b) reduction of skin pore size,
(c) improvement in skin thickness, plumpness, and/or tautness;
(d) improvement in skin suppleness and/or softness;
(e) improvement in skin tone, radiance, and/or clarity;
(f) improvement in procollagen, collagen production, and/or elastin production;
(g) improvement in maintenance and remodeling of collagen and /or elastin;
(h) improvement in skin texture and/or promotion of retexturization;
(i) improvement in skin barrier repair and/or function;
(j) improvement in appearance of skin contours;
(k) restoration of skin luster and/or brightness;
(1) replenishment of essential nutrients and/or constituents in the skin;
(m) decreased by aging and/or menopause;
(n) improvement in skin moisturization;
(o) increase in skin elasticity and/or resiliency;
(p) treatment, reduction, and/or prevention of skin sagging;
(q) reduction of pigment spots;
(s) improving the appearance of acne scars or marks;
(t) improving the appearance of stretch marks; and/or
(u) improvement in the appearance of cellulite.
In practice, the compositions of the invention are applied to skin in need of treatment. That is, skin which suffers from a deficiency or loss in any of the foregoing attributes or which would otherwise benefit from improvement in any of the foregoing skin attributes.
In certain preferred embodiments the compositions and methods of the invention are directed to the prevention, treatment, and/or amelioration of fine lines and/or wrinkles in the skin. In this case, the compositions are applied to skin in need of treatment, by which is meant skin having wrinkles and/or fine lines. Preferably, the compositions are applied directly to the fine lines and/or wrinkles. The compositions and methods are suitable for treating fine lines and/or wrinkles on any surface of the skin, including without limitation, the skin of the face, neck, and/or hands. The cosmetic compositions for treating a skin condition associated with loss of collagen and/or elastin fiber comprise, in a cosmetically acceptable vehicle, an amount of a peptide of the invention effective to enhance collagen. These collagen enhancing/stimulating agents may have the structure of formula (I) as indicated above.
The compounds of the invention and the compositions comprising said compounds are useful in the treatment of skin conditions which are ameliorated by an increased amount and/or stability of collagen and/or to accelerate recovery in said skin conditions. According to the invention, skin conditions which are ameliorated by an increased amount of collagen or skin conditions associated with loss of collagen and/or loss of collagen stability include skin wounds, therefore peptide of the inventions are useful in accelerating wound healing and in the treatment of disorders of wound healing. According to the invention, said skin conditions also include bedsores, ulcers, in particular in the elderly and debilitated, in diabetics, in patients affected by vasculopathies and coagulopathies, in patients under glucocorticoid or immunosuppressant therapies, or showing vitamin C deficiency. The general symptoms of cutaneous aging are also included within the meaning of skin conditions as used herein.
Compositions of the invention comprise one or more peptides of general formula (I) in combination with dermatologically acceptable components.
The definition "dermatologically acceptable" or "cosmetically acceptable" as utilized herein indicates that the compositions or the ingredients are suitable for topical use on human skin without risks of toxicity, incompatibility, instability, intolerance, allergic response and the like.
The term "cosmetically or dermatologically acceptable" also means compatible with the skin and/or its integuments, having a pleasant colour, odour and feel and not causing any unacceptable discomfort (stinging, tautness or redness) liable to discourage the consumer from using this composition.
Peptides of general formula (I) are formulated in compositions containing a dermatologically acceptable carrier. The dermatologically acceptable carrier include be aqueous or hydroalcoholic solution, water in oil emulsion, oil in water emulsion, microemulsion, aqueous gel, anhydrous gel, serum or vesicle dispersion. The composition of the invention may include various other and additional ingredients, which may be active, functional, conventionally used in cosmetic, personal care or topical/transdermal pharmaceutical products or otherwise. A decision to include an additional ingredient and the choice of specific additional ingredients depends on the specific application and product formulation. The CTFA Cosmetic Ingredient Handbook, 10th edition, 2004 (published by Cosmetic, Toiletry and Fragrance Association, Inc, Washinghton, D.C.) describes a wide variety of non limiting materials that can be added to the composition herein. Examples of actives which could be added include, but are not limited to, sugar amines, vitamin B3 and its derivatives, dehydroacetic acid (DHA) and its salts, phytosterols, salicylic acid compounds, ascorbic acid and derivatives, UV absorbers, keratinolytic agents, anti-oxidants, free radical scavengers, antimicrobial agents, antibacterial agents, antifungal agents, thickeners, antiperspirants and lipid thickeners.
The compositions of the invention comprise at least one dermato logically acceptable vehicle, selected on the basis of the final formulation of the product. Said vehicle is present at a concentration comprised between 50% and 99.99%, preferably between 60% and 99.9%, more preferably between 70% and 98% by weight of the composition.
The compositions of the invention are generally prepared with conventional methods known in the art for the preparations for dermatological and/or cosmetic use. Said compositions can be formulated in different physical forms, including creams, lotions, ointments, milk, gel, emulsions, suspensions, anhydrous preparations, shampoos and scalp treatment lotions, creams or lotions for care of skin or hair, sunscreens, transdermal patches, sprays, soaps, lipsticks or, in general, products for make-up.
The invention embraces the use of cosmetically acceptable (e.g., non-toxic and/or non-irritating) salts. Examples of the salts of the compounds in the present invention include salts with alkali metals such as sodium and potassium; salts with alkaline-earth metals such as calcium and magnesium; salts with amines such as monoethanolamine; salts with inorganic acids such as hydrochloric acid and sulfuric acid; and salts with organic acids such as citric acid and acetic acid. Special mention may be made of hydrochloride salts.
The cosmetic compositions according to the invention can be formulated in a variety of forms for topical application and will comprise from about 0.00001% to about 90% by weight of one or more compounds according to formula (I), and preferably will comprise from about 0.001% to about 25% by weight, and more preferably from about 0.001% to about 1% by weight. The compositions will comprise a dermatologically effective amount of the peptide of the invention, by which is meant an amount sufficient to enhance the aesthetic appearance of a given area of skin when topically applied thereto. The composition may be formulated in a variety of product forms, such as, for example, a lotion, cream, serum, spray, aerosol, cake, ointment, essence, gel, paste, patch, pencil, towelette, mask, stick, foam, elixir, concentrate, and the like, particularly for topical administration. Preferably the composition is formulated as a lotion, cream, ointment, or gel.
The compositions can include a cosmetically acceptable vehicle. Such vehicles may take the form of any known in the art suitable for application to skin and may include, but are not limited to, water; vegetable oils; mineral oils; esters such as octal palmitate, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether and dimethyl isosorbide; alcohols such as ethanol and isopropanol; fatty alcohols such as cetyl alcohol, cetearyl alcohol, stearyl alcohol and biphenyl alcohol; isoparaffins such as isooctane, isododecane and hexadecane; silicone oils such as cyclomethicone, dimethicone, dimethicone cross-polymer, polysiloxanes and their derivatives, preferably organomodified derivatives; hydrocarbon oils such as mineral oil, petrolatum, isoeicosane and polyisobutene; polyols such as propylene glycol, glycerin, butylene glycol, pentylene glycol and hexylene glycol; waxes such as beeswax and botanical waxes; or any combinations or mixtures of the foregoing.
The vehicle may comprise an aqueous phase, an oil phase, an alcohol, a silicone phase or mixtures thereof. The cosmetically acceptable vehicle may also comprise an emulsion. Non-limiting examples of suitable emulsions include water-in-oil emulsions, oil-in-water emulsions, silicone-in- water emulsions, water-in-silicone emulsions, wax-in- water emulsions, water-oil-water triple emulsions or the like having the appearance of a cream, gel or microemulsions. The emulsion may include an emulsifier, such as a nonionic, anionic or amphoteric surfactant. The oil phase of the emulsion preferably has one or more organic compounds, including emollients; humectants (such as propylene glycol and glycerin); other water- dispersible or water-soluble components including thickeners such as veegum or hydroxyalkyl cellulose; gelling agents, such as high MW polyacrylic acid, i.e. CARBOPOL 934; and mixtures thereof. The emulsion may have one or more emulsifiers capable of emulsifying the various components present in the composition. The compounds suitable for use in the oil phase include without limitation, vegetable oils; esters such as octyl palmitate, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether; fatty alcohols such as cetyl alcohol, stearyl alcohol and behenyl alcohol; isoparaffins such as isooctane, isododecane and isohexadecane; silicone oils such as dimethicones, cyclic silicones, and polysiloxanes; hydrocarbon oils such as mineral oil, petrolatum, isoeicosane and polyisobutene; natural or synthetic waxes; and the like. Suitable hydrophobic hydrocarbon oils may be saturated or unsaturated, have an aliphatic character and be straight or branched chained or contain alicyclic or aromatic rings. The oil-containing phase may be composed of a singular oil or mixtures of different oils.
Hydrocarbon oils include those having 6-20 carbon atoms, more preferably 10-16 carbon atoms. Representative hydrocarbons include decane, dodecane, tetradecane, tridecane, and C6-20- isoparaffins. Paraffinic hydrocarbons are available from Exxon under the ISOPARS trademark, and from the Permethyl Corporation. In addition, C6-2o- paraffinic hydrocarbons such as CI 2 isoparaffin (isododecane) manufactured by the Permethyl Corporation having the tradename Permethyl 99ATM are also contemplated to be suitable. Various commercially available C16 isoparaffins, such as isohexadecane (having the tradename Permethyl RTM) are also suitable. Examples of preferred volatile hydrocarbons include polydecanes such as isododecane and isodecane, including for example, Permethyl- 99 A (Presperse Inc.) and the C7-C8 through CI 2- C15 isoparaffins such as the Isopar Series available from Exxon Chemicals. A representative hydrocarbon solvent is isododecane.
The oil phase may comprise one or more waxes, including for example, rice bran wax, carnauba wax, ouricurry wax, candelilla wax, montan waxes, sugar cane waxes, ozokerite, polyethylene waxes, Fischer-Tropsch waxes, beeswax, microcrystaline wax, silicone waxes, fluorinated waxes, and any combination thereof.
Non-limiting emulsifiers included emulsifying waxes, emulsifying polyhydric alcohols, polyether polyols, polyethers, mono- or di-ester of polyols, ethylene glycol mono- stearates, glycerin mono- stearates, glycerin di-stearates, silicone-containing emulsifiers, soya sterols, fatty alcohols such as cetyl alcohol, fatty acids such as stearic acid, fatty acid salts, and mixtures thereof. The preferred emulsifiers include soya sterol, cetyl alcohol, stearic acid, emulsifying wax, and mixtures thereof. Other specific emulsifiers that can be used in the composition of the present invention include, but are not limited to, one or more of the following: sorbitan esters; poly glycery 1-3 -diisostearate; sorbitan monostearate, sorbitan tristearate, sorbitan sesquioleate, sorbitan monooleate; glycerol esters such as glycerol monostearate and glycerol monooleate; polyoxy ethylene phenols such as polyoxy ethylene octyl phenol and polyoxyethylene nonyl phenol; polyoxy ethylene ethers such as polyoxy ethylene cetyl ether and polyoxyethylene stearyl ether; polyoxyethylene glycol esters; polyoxyethylene sorbitan esters; dimethicone copolyols; polyglyceryl esters such as poly glyceryl- 3 -diisostearate; glyceryl laurate; Steareth-2, Steareth-10, and Steareth-20, to name a few. Additional emulsifiers are provided in the INCI Ingredient Dictionary and Handbook 11th Edition 2006, the disclosure of which is hereby incorporated by reference. These emulsifiers typically will be present in the composition in an amount from about 0.001% to about 10% by weight, in particular in an amount from about 0.01% to about 5% by weight, and more preferably, below 1% by weight. The oil phase may comprise one or more volatile and/or non-volatile silicone oils. Volatile silicones include cyclic and linear volatile dimethylsiloxane silicones. In one embodiment, the volatile silicones may include cyclodimethicones, including tetramer (D4), pentamer (D5), and hexamer (D6) cyclomethicones, or mixtures thereof. Particular mention may be made of the volatile cyclomethicone-hexamethyl cyclotrisiloxane, octamethyl- cyclotetrasiloxane, and decamethyl-cyclopentasiloxane. Suitable dimethicones are available from Dow Corning under the name Dow Corning 200(R) Fluid and have viscosities ranging from 0.65 to 600,000 centistokes or higher. Suitable non-polar, volatile liquid silicone oils are disclosed in U.S. Pat. No. 4,781 ,917, herein incorporated by reference in its entirety. Additional volatile silicone materials are described in Todd et al, "Volatile Silicone Fluids for Cosmetics", Cosmetics and Toiletries, 91 :27-32 (1976), herein incorporated by reference in its entirety. Linear volatile silicones generally have a viscosity of less than about 5 centistokes at 25°C, whereas the cyclic silicones have viscosities of less than about 10 centistokes at 25°C. Examples of volatile silicones of varying viscosities include Dow Corning 200, Dow Corning 244, Dow Corning 245, Dow Corning 344, and Dow Corning 345, (Dow Corning Corp.); SF-1204 and SF-1202 Silicone Fluids (G.E. Silicones), GE 7207 and 7158 (General Electric Co.); and SWS-03314 (SWS Silicones Corp.). Linear, volatile silicones include low molecular weight polydimethylsiloxane compounds such as hexamethyldisiloxane, octamethyltrisiloxane, decamethyltetrasiloxane, and dodecamethylpentasiloxane, to name a few. Non-volatile silicone oils will typically comprise polyalkylsiloxanes, polyarylsiloxanes, polyalkylarylsiloxanes, or mixtures thereof. Polydimethylsiloxanes are preferred non-volatile silicone oils. The non-volatile silicone oils will typically have a viscosity from about 10 to about 60,000 centistokes at 25°C, preferably between about 10 and about 10,000 centistokes, and more preferred still between about 10 and about 500 centistokes; and a boiling point greater than 250° C at atmospheric pressure. Non limiting examples include dimethyl polysiloxane (dimethicone), phenyl trimethicone, and diphenyldimethicone. The volatile and non-volatile silicone oils may optionally be substituted with various functional groups such as alkyl, aryl, amine groups, vinyl, hydroxyl, haloalkyl groups, alkylaryl groups, and acrylate groups, to name a few. The water-in-silicone emulsion may be emulsified with a nonionic surfactant (emulsifier) such as, for example, polydiorganosiloxane-polyoxyalkylene block copolymers, including those described in U.S. Patent No. 4, 122,029, the disclosure of which is hereby incorporated by reference. These emulsifiers generally comprise a polydiorganosiloxane backbone, typically polydimethylsiloxane, having side chains comprising -(EO)m- and/or - (PO)n- groups, where EO is ethyleneoxy and PO is 1 ,2-propyleneoxy, the side chains being typically capped or terminated with hydrogen or lower alkyl groups (e.g., CI -6, typically CI -3). Other suitable water-in-silicone emulsifiers are disclosed in U.S. Patent No. 6,685,952, the disclosure of which is hereby incorporated by reference herein. Commercially available water-in-silicone emulsifiers include those available from Dow Corning under the trade designations 3225C and 5225C FORMULATION AID; SILICONE SF-1528 available from General Electric; ABIL EM 90 and EM 97, available from Goldschmidt Chemical Corporation (Hopewell, VA); and the SILWET series of emulsifiers sold by OSI Specialties (Danbury, CT). Examples of water-in-silicone emulsifiers include, but are not limited to, dimethicone PEG 10/15 crosspolymer, dimethicone copolyol, cetyl dimethicone copolyol, PEG- 15 lauryl dimethicone crosspolymer, laurylmethicone crosspolymer, cyclomethicone and dimethicone copolyol, dimethicone copolyol (and) caprylic/capric triglycerides, polyglyceryl-4 isostearate (and) cetyl dimethicone copolyol (and) hexyl laurate, and dimethicone copolyol (and) cyclopentasiloxane. Preferred examples of water-in-silicone emulsifiers include, without limitation, PEG/PPG- 18/18 dimethicone (trade name 5225 C, Dow Corning), PEG/PPG-19/19 dimethicone (trade name BY25-337, Dow Corning), Cetyl PEG/PPG- 10/1 dimethicone (trade name Abil EM- 90, Goldschmidt Chemical Corporation), PEG-12 dimethicone (trade name SF 1288, General Electric), lauryl PEG/PPG- 18/18 methicone (trade name 5200 FORMULATION AID, Dow Corning), PEG-12 dimethicone crosspolymer (trade name 9010 and 901 1 silicone elastomer blend, Dow Corning), PEG- 10 dimethicone crosspolymer (trade name KSG-20, Shin-Etsu), and dimethicone PEG- 10/15 crosspolymer (trade name KSG-210, Shin-Etsu). The water-in-silicone emulsifiers typically will be present in the composition in an amount from about 0.001% to about 10% by weight, in particular in an amount from about 0.01% to about 5% by weight, and more preferably, below 1% by weight. The aqueous phase of the emulsion may include one or more additional solvents, including lower alcohols, such as ethanol, isopropanol, and the like. The volatile solvent may also be a cosmetically acceptable ester such as butyl acetate or ethyl acetate; or the like. The oil-containing phase will typically comprise from about 10% to about 99%, preferably from about 20% to about 85%, and more preferably from about 30% to about 70% by weight, based on the total weight of the emulsion, and the aqueous phase will typically comprise from about 1% to about 90%, preferably from about 5% to about 70%, and more preferably from about 20% to about 60% by weight of the total emulsion. The aqueous phase will typically comprise from about 25% to about 100%, more typically from about 50% to about 95% by weight water. The compositions may include liposomes. The liposomes may comprise other additives or substances and/or may be modified to more specifically reach or remain at a site following administration. Additionally, the compositions may incorporate encapsulation and/or microencapsulation technology. As is well known in the art, encapsulating materials can be selected which will release the composition upon exposure to moisture, pH change, temperature change, solubility change, or mechanical shear or rupture. Suitable encapsulating materials and methods of preparing encapsulated materials, such as spray drying, extrusion, coacervation, fluidized bed coating, liposome entrapment and others, are disclosed in, for example, U.S. Patent Application Publication No. 2005/0000531 to Shi; Uhlmann, et al, "Flavor encapsulation technologies: an overview including recent developments" Perfumer and Flavorist, 27, 52-61 , 2002; and "Selection of Coating and Microencapsulation Processes" by Robert E. Sparks and Irwin Jacobs in Controlled-Release Delivery Systems for Pesticides, Herbert B. Scher ed., Marcel Dekker, New York, N.Y., 1999, pp. 3-29, the contents of which are hereby incorporated by reference.
The compositions incorporating encapsulation and/or microencapsulation technology may form nanoparticles. The term "nanoparticle" as used herein refers to a nanometer-sized particle, having a diameter of between about 1 nanometer and about 999 nanometers; the term "nanoparticles" as used herein refers to nanometer-sized particles, nanoclusters, clusters, particles, small particles, and nanostructured materials. The composition may optionally comprise other cosmetic actives and excipients, obvious to those skilled in the art including, but not limited to, fillers, emulsifying agents, antioxidants, surfactants, film formers, chelating agents, gelling agents, thickeners, emollients, humectants, moisturizers, vitamins, minerals, viscosity and/or rheology modifiers, sunscreens, keratolyses, depigmenting agents, retinoids, hormonal compounds, alpha- hydroxy acids, alpha-keto acids, anti-mycobacterial agents, antifungal agents, antimicrobials, antivirals, analgesics, lipidic compounds, anti-allergenic agents, HI or H2 antihistamines, anti-inflammatory agents, anti-irritants, antineoplastics, immune system boosting agents, immune system suppressing agents, anti-acne agents, anesthetics, antiseptics, insect repellents, skin cooling compounds, skin protectants, skin penetration enhancers, exfollients, lubricants, fragrances, colorants, depigmenting agents, hypopigmenting agents, preservatives, stabilizers, photostabilizing agents, sunscreens, and mixtures thereof. In addition to the foregoing, the cosmetic compositions of the invention may contain any other compound for the treatment of skin disorders. Such additional actives and/or excipients may work in concert with the compositions of the current invention to achieve cumulative or synergistic improvements in the aesthetic appearance.
Colorants may include, for example, organic and inorganic pigments and pearlescent agents. Suitable inorganic pigments include, but are not limited to, titanium oxide, zirconium oxide and cerium oxide, as well as zinc oxide, iron oxide, chromium oxide and ferric blue. Suitable organic pigments include barium, strontium, calcium, and aluminium lakes and carbon black. Suitable pearlescent agents include mica coated with titanium oxide, with iron oxide, or with natural pigment.
Various fillers and additional components may be added. Fillers are normally present in an amount of about 0 weight % to about 20 weight %, based on the total weight of the composition, preferably about 0.1 weight % to about 10 weight %. Suitable fillers include without limitation silica, treated silica, talc, zinc stearate, mica, kaolin, Nylon powders such as Orgasol(TM), polyethylene powder, Tefion(TM), starch, boron nitride, copolymer microspheres such as Expancel(TM) (Nobel Industries), Polytrap(TM) (Dow Corning) and silicone resin microbeads (Tospearl(TM) from Toshiba), and the like. In one embodiment of the invention, the compositions may include additional skin actives such as, but not limited to, botanicals, keratolytic agents, desquamating agents, keratinocyte proliferation enhancers, collagenase inhibitors, elastase inhibitors, depigmenting agents, anti-inflammatory agents, steroids, anti-acne agents, antioxidants, salicylic acid or salicylates, thiodipropionic acid or esters thereof, and advanced glycation end-product (AGE) inhibitors to achieve cumulative or synergistic improvements in the aesthetic appearance of the treated skin. In a specific embodiment, the composition may comprise at least one additional botanical, such as, for example, a botanical extract, an essential oil, or the plant itself. Suitable botanicals include, without limitation, extracts from Abies pindrow, Acacia catechu, Aiisma orientate, Aloe, Amorphophallus campanulatus, Anogeissus iatifoiia, Asmunda japonica, Azadirachta indica, Butea frondosa, Butea monosperma, Cedrus deodara, Chamomile, Derris scandens, Portulaca oleracea, Eclipta prostrala, Emblica officinalis, Erythina indica, Ficus benghaiensis, Glycyrrhiza glabra, Humilus scandens, Ilex purpurea Hassk, Innula racemosa, Ixora chinensis, Ligusticum chiangxiong, Ligusticum lucidum, Mallotus philippinensis, Medemia noblis, Melicope hayesii, Mimusops elengi, Morinda citrifolia, Moringa oleifera, Naringi crenulata, Nerium indicum, Piper betel, Portulaca oleracea, Pouzolzia petandra, Psoralea corylifolia, Rhinacanthus nasutus, Sapindus rarek, Sesbania grandiflora, Stenoloma chusana, Terminalia bellerica, Tiliacora triandra, tomato glycolipid and mixtures thereof. The composition may comprise additional active ingredients having anti-aging benefits, as it is contemplated that synergistic improvements may be obtained with such combinations. Exemplary anti-aging components include, without limitation, botanicals (e.g., Butea frondosa extract); phytol, thiodipropionic acid (TDPA) and esters thereof; retinoids (e.g., all-trans retinoic acid, 9-cis retinoic acid, phytanic acid and others); hydroxy acids (including alpha-hydroxyacids and beta- hydroxyacids), salicylic acid and salicylates; exfoliating agents (e.g., glycolic acid, 3,6,9- trioxaundecanedioic acid, etc.), estrogen synthetase stimulating compounds (e.g., caffeine and derivatives); compounds capable of inhibiting 5 alpha-reductase activity (e.g., linolenic acid, linoleic acid, finasteride, and mixtures thereof); barrier function enhancing agents (e.g., ceramides, glycerides, cholesterol and its esters, alpha-hydroxy and omega-hydroxy fatty acids and esters thereof, etc.); collagenase inhibitors; and elastase inhibitors; to name a few. Exemplary retinoids include, without limitation, retinoic acid (e.g., all-trans or 13-cis) and derivatives thereof, retinol (Vitamin A) and esters thereof, such as retinol palmitate, retinol acetate and retinol propionate, and salts thereof. In another embodiment, the topical compositions of the present invention may also include one or more of the following: a skin penetration enhancer, an emollient, a skin plumper, an optical diffuser, a sunscreen, an exfoliating agent, and an antioxidant. An emollient provides the functional benefits of enhancing skin smoothness and reducing the appearance of fine lines and coarse wrinkles. Examples include isopropyl myristate, petrolatum, isopropyl lanolate, silicones (e.g., methicone, dimethicone), oils, mineral oils, fatty acid esters, or any mixtures thereof. The emollient may be preferably present from about 0.1 wt % to about 50 wt% of the total weight of the composition.
A skin plumper serves as a collagen enhancer to the skin. An example of a suitable, and preferred, skin plumper is palmitoyl oligopeptide. Other skin plumpers are collagen and/or other glycosaminoglycan (GAG) enhancing agents. When present, the skin plumper may comprise from about 0.1 wt % to about 20 wt% of the total weight of the composition. An optical diffuser is a particle that changes the surface optometries of skin, resulting in a visual blurring and softening of, for example, lines and wrinkles. Examples of optical diffusers that can be used in the present invention include, but are not limited to, boron nitride, mica, nylon, polymethylmethacrylate (PMMA), polyurethane powder, sericite, silica, silicone powder, talc, Teflon, titanium dioxide, zinc oxide, or any mixtures thereof. When present, the optical diffuser may be present from about 0.01 wt % to about 20 wt% of the total weight of the composition. A sunscreen for protecting the skin from damaging ultraviolet rays may also be included. Preferred sunscreens are those with a broad range of UVB and UVA protection, such as octocrylene, avobenzone (Parsol 1789), octyl methoxycinnamate, octyl salicylate, oxybenzone, homosylate, benzophenone, camphor derivatives, zinc oxide, and titanium dioxide. When present, the sunscreen may comprise from about 0.01 wt % to about 70 wt % of the composition.
Suitable exfoliating agents include, for example, alpha-hydroxyacids, beta- hydroxyacids, oxaacids, oxadiacids, and their derivatives such as esters, anhydrides and salts thereof. Suitable hydroxy acids include, for example, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid and derivatives thereof. A preferred exfoliating agent is glycolic acid. When present, the exfoliating agent may comprise from about 0.1 wt % to about 80 wt % of the composition. An antioxidant functions, among other things, to scavenge free radicals from skin to protect the skin from environmental aggressors. Examples of antioxidants that may be used in the present compositions include compounds having phenolic hydroxy functions, such as ascorbic acid and its derivatives/esters; beta-carotene; catechins; curcumin; ferulic acid derivatives (e.g. ethyl ferulate, sodium ferulate); gallic acid derivatives (e.g., propyl gallate); lycopene; reductic acid; rosmarinic acid; tannic acid; tetrahydrocurcumin; tocopherol and its derivatives; uric acid; or any mixtures thereof. Other suitable antioxidants are those that have one or more thiol functions (-SH), in either reduced or non-reduced form, such as glutathione, lipoic acid, thioglycolic acid, and other sulfhydryl compounds. The antioxidant may be inorganic, such as bisulfites, metabisulfites, sulfites, or other inorganic salts and acids containing sulfur. Compositions of the present invention may comprise an antioxidant preferably from about 0.001 wt % to about 10 wt%, and more preferably from about 0.01 wt% to about 5 wt%, of the total weight of the composition. Other conventional additives include: vitamins, such as tocopherol and ascorbic acid; vitamin derivatives such as ascorbyl monopalmitate; thickeners such as hydroxyalkyl cellulose; gelling agents; structuring agents such as bentonite, smectite, magnesium aluminum silicate and lithium magnesium silicate; metal chelating agents such as EDTA; pigments such as zinc oxide and titanium dioxide; colorants; emollients; and humectants.
The invention provides a method for improving aging skin by topically applying a composition comprising the peptide of the invention, preferably in a cosmetically acceptable vehicle, over the affected area for a period of time sufficient to reduce, ameliorate, reverse or prevent dermatological signs of aging. This method is particularly useful for treating signs of skin photoaging and intrinsic aging. Generally, the improvement in the condition and/or aesthetic appearance is selected from the group consisting of: reducing dermatological signs of chronological aging, photo-aging, hormonal aging, and/or actinic aging; preventing and/or reducing the appearance of lines and/or wrinkles; reducing the noticeability of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, perpendicular wrinkles between the eyes, horizontal wrinkles above the eyes, and around the mouth, marionette lines, and particularly deep wrinkles or creases; preventing, reducing, and/or diminishing the appearance and/or depth of lines and/or wrinkles; improving the appearance of suborbital lines and/or periorbital lines; reducing the appearance of crow's feet; rejuvenating and/or revitalizing skin, particularly aging skin; reducing skin fragility; preventing and/or reversing of loss of glycosaminoglycans and/or collagen; ameliorating the effects of estrogen imbalance; preventing skin atrophy; preventing, reducing, and/or treating hyperpigmentation; minimizing skin discoloration; improving skin tone, radiance, clarity and/or tautness; preventing, reducing, and/or ameliorating skin sagging; improving skin firmness, plumpness, suppleness and/or softness; improving procollagen and/or collagen production; improving skin texture and/or promoting retexturization; improving skin barrier repair and/or function; improving the appearance of skin contours; restoring skin luster and/or brightness; minimizing dermatological signs of fatigue and/or stress; resisting environmental stress; replenishing ingredients in the skin decreased by aging and/or menopause; improving communication among skin cells; increasing cell proliferation and/or multiplication; increasing skin cell metabolism decreased by aging and/or menopause; retarding cellular aging; improving skin moisturization; enhancing skin thickness; increasing skin elasticity and/or resiliency; enhancing exfoliation; improving microcirculation; decreasing and/or preventing cellulite formation, e.g., on the thighs; alleviating the appearance and formation of stretch marks; and any combinations thereof. Without wishing to be bound by any particular theory, it is believed that the compositions of the present invention enhance and improve the aesthetic appearance of skin by stimulation of collagen and restoring and maintaining homeostasis of this component. The composition will typically be applied to the skin one, two, or three times daily for as long as is necessary to achieve desired anti-aging or therapeutic benefit. The treatment regimen may comprise daily application for at least one week, at least two weeks, at least four weeks, at least eight weeks, or at least twelve weeks. Chronic treatment regimens are also contemplated. In further embodiments, trans dermal patches including single-layer, multilayer, reservoir, matrix patches and the like in the form of nose strips, peel-off paper masks, specialized treatment patches for eyes and facial contours, etc.; bandages including gauze, tube, and the like; poultices such as those used as facial masks, etc. are used to administer the cosmetic compositions of the current invention to areas of skin in need thereof for extended periods of time. The peptide of the invention as active component is topically applied to an "individual in need thereof," by which is meant an individual that stands to benefit from reducing visible signs of skin damage or aging. In a specific embodiment, the peptide component is provided in a physiologically, cosmetically, and dermatologically-acceptable vehicle, diluent, or carrier, where the composition is topically applied to an affected area of skin and left to remain on the affected area in an amount effective for improving the condition and aesthetic appearance of skin. In one embodiment, methods for treating fine lines and wrinkles comprise topically applying the inventive peptide and compositions of the invention to the skin of an individual in need thereof, e.g., topical application directly to the fine line and/or wrinkle in an amount and for a time sufficient to reduce the severity of the fine lines and/or wrinkles or to prevent or inhibit the formation of new fine lines and/or wrinkles. The effect of a composition on the formation or appearance of fine lines and wrinkles can be evaluated qualitatively, e.g., by visual inspection, or quantitatively, e.g., by microscopic or computer assisted measurements of wrinkle morphology (e.g., the number, depth, length, area, volume and/or width of wrinkles per unit area of skin). This embodiment includes treatment of wrinkles on the skin of the hands, arms, legs, neck, chest, and face, including the forehead. It is also contemplated that the compositions of the invention will be useful for treating thin skin by topically applying the composition to thin skin of an individual in need thereof. "Thin skin" is intended to include skin that is thinned due to chronological aging, menopause, or photo-damage. In some embodiments, the treatment is for thin skin in men, whereas other embodiments treat thin skin in women, premenopausal or post-menopausal, as it is believed that skin thins differently with age in men and women, and in particular in women at different stages of life. The method of the invention may be employed prophylactically to forestall aging including in subjects that have not manifested signs of skin aging, most commonly in individuals under 25 years of age. The method may also reverse or treat signs of aging once manifested as is common in subjects over 25 years of age. The composition according to the invention may be in any galenical form conventionally used for topical application and especially in the form of aqueous gels, or aqueous or aqueous-alcoholic solutions. By adding a fatty or oily phase, it may also be in the form of dispersions of lotion type, emulsions of liquid or semi- liquid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous phase (O/W) or conversely (W/O), or suspensions or emulsions of soft, semisolid or solid consistency of the cream or gel type, or alternatively multiple emulsions (W/O/W or 0/W/O), microemulsions, vesicular dispersions of ionic and/or nonionic type, or wax/aqueous phase dispersions. These compositions are prepared according to the usual methods. The invention also relates to compositions conditioned in pressurized form in an aerosol device or in a pump-dispenser bottle; conditioned in a device equipped with a perforated wall, especially a grille; conditioned in a device equipped with a ball applicator ("roll-on"); characterized in that they contain at least perlite particles. In this regard, they contain the ingredients generally used in products of this type, which are well known to those skilled in the art. "Physiologically or cosmetically or dermatologically acceptable medium or vehicle or carrier" is also understood to mean a medium compatible with the materials and / or human keratin fibers such as, but not limited to, skin, mucous membranes, nails, scalp and / or hair.
This physiologically acceptable medium comprises water, optionally in admixture or not with one or more organic solvents such as alcohols, in particular ethanol, isopropanol, tert-butanol, n- butanol, polyols such as glycerol, propylene glycol, butylene glycol and polyol ethers.
The composition according to the invention may also comprise a fatty phase, which may include oils, gums, waxes commonly used in the field of application considered.
The cosmetic compositions according to the invention may contain active ingredients, additives or excipients customary in cosmetics: biological assets (Anti-aging, fat skinning, lightening, whitening, anti-perspirant, anti-oxidant, etc.), pigments, dyes, sunscreens, polymers, film- forming, oils, fats, silicones, moisturizers, emollients, solvents, surfactants, vitamins, and / or preservatives, or other cosmetic ingredients.
The compositions of the invention may be in the form of gels, sera, of direct emulsions, reverse or multiple sticks, poured hot, free or compacted powder, or cream.
The composition according to the invention may be in any pharmaceutical form normally used in the cosmetic and dermatological fields.
It can be especially in the form of an aqueous, aqueous-alcoholic solution, optionally gelled, a dispersion of the lotion type biphasic optionally, an oil-in-water or multiple water-in-oil emulsion, an aqueous gel, an oil dispersion (s) in an aqueous phase, in particular using spherules, these spherules to be better or polymeric particles, lipid vesicles of ionic and / or nonionic, or alternatively in the form of a powder, a serum, a paste or a flexible rod.
It can be solid consistency, pasty or liquid more or less fluid.
Advantageously, the combination is administered orally, topically or intra-dermal. According to a particular embodiment, the composition comprises a physiologically acceptable medium for topical, oral administration or by injection, in particular intra-dermal.
Alternatively, the composition is in the form of a cosmetic cream.
In another embodiment, the composition is injectable and comprises an injection medium, such as one or more macromolecules of the extracellular matrix, such as hyaluronic acid, collagen, and / or cells, and / or extracted from adipose tissue or preparation containing.
The composition of the invention may therefore be suitable for injection intra-skin by use of a syringe, using one or more micro-needles, or an implant or a biodegradable reservoir.
The term "bio-degradable container" indicates a device comprising degradable molecules in a more or less long time, often a few weeks to several months or even years after introduction into the skin.
For these types of compositions can be used the active ingredients of the invention in combination with a extra-cellular medium injection such that one or more of the matrix macromolecules such as hyaluronic acid or collagen, or cells adipose tissue extracts or preparations containing it.
In general, extracts of adipose tissue from the body of the subject receiving the injection of the composition of the invention, but may be otherwise.
This extract can be from another subject or group of subjects.
The invention further relates to an injection device prefilled with the combination or composition according to the invention intra-cutaneous.
The invention also relates to an intra-dermal injection kit comprising an intra-dermal injection device, such as a syringe, using one or more micro-needles, an implant or a biodegradable container, said kit comprising also the composition physically separated according to the particular injection device for filling injection just before the injection device invention.
The invention also relates to the cosmetic use (for aesthetic purposes) of the peptides of the invention for preventing and / or treating the signs of skin aging and / or photo-aging, in particular to reduce ptosis, reduce the loss of skin firmness, reduce visibility of wrinkles or skin wrinkles, or improve the quality and / or the transparency of the skin.
In this aspect of the invention, the use is exclusively aesthetic treatment of a subject, preferably human, and therefore excludes any surgical treatment, or therapy.
In this aspect, the invention is ultimately decreasing the visibility of wrinkles, thus justifying the use of this aesthetic goal. Means reduce the visibility of wrinkles: reducing the visibility or the depth of the grooves formed at the wrinkles of the skin surface, visually assessed by a human or by means of a suitable apparatus to quantify the appearance of skin wrinkles.
The present invention also provides a method of cosmetic care for immediate filling wrinkles, characterized in that it comprises the topical application, oral administration or intra-dermal injection of a composition according to the invention.
This topical or intra-dermal injection is performed at the zone to be filled, or nearby.
In general, this application or injection at least 2 cm, and preferably less than 1 cm, parallel to the filling lines is performed.
Preferably, the composition of the invention is an emulsified cosmetic composition comprising a water and an oil phase, and dispersed in the water an oil phases (a) at least one peptide of general formula (I), (b) optionally an additional anti-aging agent retarding the effects of natural aging and free radical activity on the skin's complexion, and (c) a sunscreen retarding the effects of aging due to sunlight on the skin's complexion. The emulsified cosmetic composition further comprises (d) a preservative for preventing microbial growth in the composition, (e) a thickener to increase the viscosity of the composition, (f) an antioxidant to prevent rancidity and discoloration of the composition, and (g) an emulsifier for combining the water phase and oil phase ingredients as well as for giving the composition a suitable texture when applied to the skin.
The anti-aging agent is selected from the group consisting of proteins, flavonoid compounds, glycogen, and combination thereof. The proteins can comprise serum proteins and hydrolyzed animal proteins. The flavonoid compounds can be provided by extracts of the plants butcher broom, buckwheat, and passion flower. The sunscreen can be selected from the group consisting of paraminobenzoic acid derivatives, oxyphenones, benzophenones, titanium dioxide, zinc oxide, cinnamate, and combinations thereof. The preservatives can be propylene glycol, trisodium EDTA, Phenonip , a preservation complex, and combinations thereof. The preservation complex contains methylparaben, propylparaben, 2-bromo-2-nitropropane-l,3diol, and hexamidine diisethionate. Phenonip is a commercially available preservative and contains phenoxyethanol, methylparaben, ethylparaben, propylparaben, and butylparaben. The antioxidant can be Tenox II and/or ascorbyl palmitate. Tenox II is a commercial product which contains propylene glycol, butylated hydroxyanisole, propyl gallate, and citric acid. Preferred thickeners are xanthan gum and carrageenan. The composition can also contain one or more colorants, such as titanium dioxide. Anti-aging ingredients
The agents for retarding the effects of natural aging and aging due to free radical activity on the skin's complexion are selected from the group consisting of proteins, flavonoid compounds, glycogen, and combinations thereof. The emulsified composition of the present invention employs a commercial source for the proteins, flavonoid compounds, and glycogen. These ingredients are supplied in various combinations in two products, called Flavoplasmine LS2704 P2 (hereinafter Flavoplasmine) and Iconoderm LS 1054 B (hereinafter Iconoderm), both available from Lab Serobiologiques, Inc., Somerville, New Jersey.
The active ingredients of Flavoplasmine are serum proteins, hydro lyzed animal proteins and flavonoid compounds. The flavonoid compounds within the Flavoplasmine are furnished by butcher broom extract, buckwheat extract, and passion flower extract. The Flavoplasmine further contains a mono stearatepoly ethylene glycol ester of stearic acid (PEG 6-32) serving as an emulsifier, and glycerin functioning as a solvent.
Iconoderm comprises serum proteins, hydrolyzed animal proteins, and glycogen. Sodium lactate is present for buffering. Iconoderm further contains sodium pyrrolidone carboxylate. The anti-free radical ingredients of the emulsified cosmetic composition can be selected from the group consisting of derivatives of vitamins C or E, selenium metal compounds, or beta carotene derivatives. Sunscreens
The agents protecting against aging of the skin's complexion due to sunlight are one or more of the sunscreens identified in the following table.
SUNSCREEN INGREDIENTS
Aminobcnzoic Acid
Cifiojate
Piftteealtmittt p*M<tthoxycinn«fiiite
KgaJioyt Trioleate
Eihyl 4*{ is{f]ydroxy propyl] Atnhwbeszoitc
2-EihyIhexyt 2>Cyano ,34ip enylacrylm
Ethy!bexyl -p-wtfl«fcii»»et*te
2-Ethyhexyl Selicylite
Glyceryl AmiisobetttOftte
Hemosatitfe
Lawyxtc with Dyh} troxyacetone
Mcnthyl AMlffaniiste
Oxybenzone
Pudirruie A
Figure imgf000032_0001
2>Fbeny aniumki»oir-5-Sttifonic Acid
Red Pciro tum
S»§setwiie»e
Titanium Dioxide
TriethanoUminf Sa i y tc
A satisfactory cosmetic composition comprises octyldimethyl paraminobenzoic acid and benzophenone-3. Preservatives
Since the emulsified cosmetic composition is manufactured under clean but non-sterile conditions, preservatives are used to prevent the growth of microbes. A sufficient quantity of one or more preservatives is added so that the emulsified cosmetic composition withstands the growth of bacteria from an experimental inoculation for at least three months. The emulsified composition can be prepared with propylene glycol, trisodium EDTA, a commercially obtained composition known as Phenonip , and a preservation complex. The preservation complex is a white, colorless, crystalline powder. It contains methylparaben, propylparaben, 2-Bromo-2-Nitropropane-l ,3-Diol, and hexamidine isethionate. Phenonip is a practically colorless viscous liquid mixture of phenoxyethanol, methylparaben, ethylparaben, propylparaben, and butylparaben available from Nipa Laboratories, Inc., Wilmington, Del.
Antioxidants
Because the emulsified cosmetic composition is applied to the skin, it is essential to a consumer that the composition's appearance and odor be pleasing. To maintain the composition's color and prevent malodorous developments, an antioxidant may be included in the cosmetic composition. Two antioxidants can be used in the cosmetic composition: Tenox II and ascorbyl palmitate. Tenox II is obtained from Eastman Chemical Products, Inc., Kingsport, Tenn. and contains propylene glycol, butylated hydroxyanisole, propyl gallate, and citric acid.
Emulsifiers
Emulsifiers serve two functions in the present invention. They act like an emulsifier to combine the water soluble and non-water soluble phases together; that is, form a stable bridge between the waters and the oils of the ingredients. The emulsifiers also serve as emollients, providing a pleasant, aesthetically appropriate, tactile feeling when the emulsified composition is applied to the skin. The preferred cosmetic composition comprises the preferred emulsifiers namely isodecyl oleate, CI 2- 15 alcohols benzoate, arachidyl propionate, octyldodecyl stearoyl stearate, steareth-2, PEG-8 stearate, isoceteth-20, steareth-21 , acetylated lanolin, dimethicone and 3-cyclohexene-l- methanol .alpha., 4-dimethyl-.alpha.(4-methyl-3-pentenyl) (hereinafter "bisabolol").
Thickener
Preferably the composition contains sufficient thickener that the cosmetic composition does not run off the face and other skin areas when applied. The thickeners complement the function of the emulsifiers in holding together the water and oil phases of the composition. The emulsified composition preferably employs as thickeners xanthan gum and carrageenan.
Emulsified base
Demineralized water is preferably used in the cosmetic composition as the emulsified base. Colorant
The cosmetic composition can include at least one colorant, preferably titanium dioxide.
MATERIAL AND METHODS
Chemical synthesis of peptides
Abbreviations used in the description of the chemistry are: Boc: tert-butoxycarbonyl; DIPC: diisopropyl carbodiimide; DIPEA: diisopropylamine; DMF: dimethyl formamide; Fmoc: fluorenylmethyloxycarbonyl; HATU: 2(l-[Bis(dimethylamino)methylene]-lH-l ,2,3-triazolo [4,5- b]pyridinium 3-oxid hexafluorophosphate); HO At: 1 -hydro xy-7-azabenzotriazole; PEG: polyethylene glycol; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu: tert-butyl; Trt: trityl.
Peptides of general formula (I) were prepared by Fmoc/tBu methodologies using a polystyrene resin (PS) or PEG-PS resin derivatized with Rink amide for the production of carboxyamide peptides at the C-terminus. The synthetic strategy is based on the elongation of the peptide starting from the C-terminus, through sequences of deprotection of the a-amino group and acylation with the activated carboxylic group of the subsequent amino acid. The a-amino groups protected as Fmoc, while the side chains are protected, when required, with acid labile protecting groups, such as Boc for lysine (Lys(Boc)), ornitine (Orn(Boc)), nor-arginine (Agb(Boc)), Pbf for arginine (Arg(Pbf)), Trt for histidine (His(Trt)) and asparagine (Asn(Trt), t-butyl for aspartic acid (Asp(tBu)), glutamic acid (Glu(tBu)) and hydroxyproline (Hyp(tBu)). All synthesis were carried out in an automated peptide synthesizer enabling incorporation of aminoacids every 60 minutes, via activation of the single amino acids HATU in the presence of DIPEA in DMF.
At the end of the synthesis, when required, the acetyl, palmitoyl or other acyl groups were introduced by conventional chemistry, for example by reaction with anhydrides or by coupling in the presence of HO At and DIPC in DMF. The completion of the reaction was followed by using the Kaiser test in the presence of ninhydrin to evaluate the presence of free amino groups.
The cleavage of peptides from the resins and the concomitant removal of protecting groups from the side chains of the single aminoacids was carried out by treating with a cleavage mixture trifluoroacetic acid: H20:phenol: triisopropylsilane = 87.5:5:5:2.5 for 60-90 minutes, depending on the protecting groups. The peptides precipitated from cold tert-butyl-methyl ether and were centrifuged at 3600rpm for 30 min at 4°C. The precipitated peptide was washed 3 times and finally freeze dried with a mixture of H20/CH3CN containing 0.1% trifluoroacetic acid.
All peptides were purified by reverse phase high pressure liquid chromatography (RP-HPLC) using the WATERS 2489 chromatographic pump or alternatively the WATERS 600/E coupled with a detector WATERS 2545 or alternatively a JASCO UV-975 detector, using chromatographic columns X-Bridge BEH130 Perp C18 ΙΟμιη (19x150 mm) or X-Bridge BEH130 Perp C18 5μιη (30x150 mm). All the peptides were characterized before and after purification by using a Waters UPLC/MS ACQUITY system equipped with mass spectrometer Waters SQ. All peptides were obtained, after liophilization, with purity > 95%. The exchange of the trifiuoroacetate counterion depends on the selected counter ion (acetate, chloride, etc). For example, the preparation of peptides with acetate as counter ion was done through ionic exchange on resin ToyoPeral DEAE 650C, by conditioning the resin with acetic acid and eluting the peptides preloaded in water with a 40 fold excess of resin/DEAE with respect to the peptide equivalents. The peptides as eluted are directly freeze dried.
The preparation of peptides as chlorohydrates was carried out by solubilizing the freeze dried peptides with a 1 mM solution of HC1 followed by liophilization. This operation can be repeated more than ones: completion of the exchange is followed by F19-NMR analysis.
Liophilized peptides are stored at -20°C.
The peptides prepared according to the above methodology are described in Table 1.
Table 1
Peptide Molecular Weight
M+l
Ac-PHREN 693.71
Palm-PHREN 890.08
Ac-PHREN-K-Palm 1060.29
Ac-PHRE 579.61
Ac-PHREQ 707.74
Ac-Hyp-HREN 709.71
Ac-Tic-HREN 755.78
Ac-Azt-HREN 679.68
Ac-P-Tha-REN 709.77
HREN 554.56
Ac-PHEN 537.52
Ac-PREN 556.57
Ac-Pro-HREN 693.71
Ac-hPro-HREN 707.74
Ac-P-Thz-REN 710.76
Ac-PH-Orn-EN 651.68
Ac-PHRDN 679.68 Ac-PHKEN 665.70
Ac-PH-Agb-EN 679.30
Collagen I induction assay in human fibroblasts
Assay system
Normal human skin fibroblasts from two different lots, deriving from adult derma, were obtained by Lonza. Cells were cultured in Fibroblast Growth Media (FGM-2 Bullet Kit) and used between passages P2-P6. The day before the assay, cells were plated (10,000 cells/well) in 96 wells plates ^Clear, Greiner) in Dulbecco' Modified Eagle's Medium (DMEM) containing 5% fetal bovine serum, 100 Ul/ml penicilline, 100 μg/ml di streptomycine, 1% ascorbic acid. The cells were incubated for 72 hours with different dosages of peptides.
Serial dilutions of the peptides were prepared in DMSO and diluted directly in the assay in order to have a final percentage of DMSO of 0.1.
After removal of the assay medium, cells were washed 3 times with Phosphate -buffered saline (PBS) and the monolayer was fixed by 15 min incubation with ethanol at 95%. After 3 further washing with PBS containing 1% Triton X-100, cells were incubated for two hours in PBS supplemented with 1 % bovine serum albumin and for additional two hours with a monoclonal murine antibody, specific for collagen type I (Ab 90395, Abeam) diluted 1 :1000 (v/v). After three additional washes with PBS containing 1% Triton X-100, a secondary antibody conjugated with the fiuorophore Alexafluor 488 (1 :3000) and the nuclear staining Hoechst were added and incubated for two hours in PBS containing 1% bovine albumin. After three washes with PBS and 1% Triton X-100, 50 μΐ/well of PBS were added and the plate was analyzed with throughput laser scanner imager (Acumen eX3 TTP Labtech, IN Cell Analyzer 1000 GE Healthcare).
Negative controls are carried out under the same conditions but in the absence of the peptide.
Analysis of results
Samples were analyzed on microplates by automated fluorescence microscopy. Two techniques were employed:
cytometry to detect most efficacious compounds;
fluorescence microscopy for the quantification and visualization of the increased amount of collagen following the treatment. For plate based cytometry an Acumen Explorer (TTP Labtech Ltd, UK) plate reader was used. When the signal associated to one pixel exceeds the average two times the standard deviation the object is identified in the microplate wells. The light emitted from the samples is recorded through a photomultiplier. Sample scan was done with two laser sources. The first, having wavelength of 408 nM, causes the excitation of the nuclear marker Hoechst 33348 (Life Technologies, US). The light emitted by said marker, registered with an emission filter 460 nM, allows for the quantification of the number of nucleus. Said value is used to compensate the fluctuations of signals due to change in the total cell amount within the samples. The signals of the immunostained collagen type I were obtained by laser excitation at 488 nM and an excitation filter at 540 nM. Only object with dimensions greater than average were considered as cells. The sum of signals from single objects for each sample was normalized to the nucleus count described before. The so obtained value represents, in relative fluorescence units per cells, the induction of collagen type I due to treatment.
Fluorescence microscopy was used to quantify and localize within the cell the induced collagen type I. Toward this end the instrument InCell 1000 (GE Healthcare, USA) was used. Fluoro fores excitation, both nuclear and from immunostained collagen, was done with a Xenon lamp. The incoming and outgoing light was filtered by using a set of filters similar to that used for the cytometer. For each sample ten images per two channels were acquired via CCD camera: one originated from the excitation of the nuclear marker and one originated from the excitation of the fluorophores used for the immuno staining of collagen type I. The quantification of the latter was done by image analysis (lOx phase objective) with the InCell Investigator software (GE Healthcare, USA). The signal related to collagen type I is localized within cytoplasmic vesicles, probably due to the fact that this protein is secreted. The analysis protocol is as follows:
segmentation of nuclei by using the tool provided by the software;
- segmentation of the vesicles containing collagen type I by using the tool provided by the software;
association of the vesicles with the corresponding expanded nuclei by superimposition of the images;
quantification of the immuno staining signal of collagen type I associated to each cell.
The average of signals for each sample represents the induction level of collagen type I per cell which is triggered by the treatment. RESULTS
The results obtained are reported in Table 2. The induction of collagen type I is measured by plate cytometry as described in the Material and Method section.
Table 2
Figure imgf000038_0001
The results described in Table 2 clearly demonstrate that peptides of the invention significantly increase the amount of Collagen I when tested at concentrations comprised between 0.3μΜ and 30μΜ. Even at the lower concentration of 0.3μΜ of peptides AcPHREN, AcPHRE, HREN, AcPREN the synthesis of collagen is augmented by, respectively, 1.4, 2.0, 2.0 and 1.8 fold relative to the blank tests (without peptide). Under the same conditions, the reference prior art peptides PalmGQPR, PalmGHK, AcGHK increase collagen amount by 1.6, 1.8, 1.4 fold. Therefore, experimental data provided herein demonstrate that the claimed peptides have a biological activity comparable or higher than that of other peptides already known in the art to increase collagen in human fibroblasts.
The good results obtained in these experiments clearly indicate that peptides of the invention and compositions comprising said peptides act at the level of collagen type I synthesis and are therefore particularly useful for accelerating the process of cutaneous regeneration and for restoring improved structure to the skin fibers network.

Claims

1. A peptide of general formula (I)
Figure imgf000040_0001
m, n, p, q = 0, 1 and if one of m, n, p o q is 0, the others are 1 ;
y = 0, l , 2; z = 0, 1;
Ri = H, OH;
A = H, R2CO wherein R2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
X is selected from an amino acid, an amino acid derivative, OR3, NR3R4 wherein R3 and R4 are each independently selected from H, C1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof,
with the exclusion of the compounds wherein:
Ri = H; m = n = p = q = l; y = l; z = 0; X = NH2.
2. The peptide according to claim 1 wherein A is selected from acetyl or palmitoyl.
3. The peptide according to claim 1 selected in the group consisting of:
- Acetyl-Pro-His-Arg-Glu-Asn-Lys-(N8-Palmitoyl) (SEQ ID No. 1);
- Acetyl-Pro-His-Arg-Glu-Gln (SEQ ID No. 2);
- Acetyl-Pro-His-Arg-Glu;
- His-Arg-Glu-Asn;
- Acetyl-Pro-Arg-Glu-Asn;
- Acetyl-Hyp-His-Arg-Glu-Asn (SEQ ID No. 3);
- Acetyl-Pro-His-Glu-Asn.
4. The peptide of general formula (I),
Figure imgf000041_0001
m, n, , q = 0, 1 e and if one of m, n, p o q is 0, the others are 1;
y = 0, l , 2; z = 0, 1;
Ri = H, OH;
A = H, R2CO wherein R2 is an alkyl group containing 1 to 22 carbon atoms, linear, branched or cyclic, saturated or unsaturated, hydroxylated or non hydroxylated, sulfurated or non-sulfurated, oxygenated or non-oxygenated;
X is selected from a amino acid, an amino acid derivative, OR3, NR3R4 wherein R3 and R4 are each independently selected from H, C1-22 alkyl linear or branched, saturated or unsaturated; pharmaceutically acceptable salts, isomers, stereoisomers and tautomers thereof, for medical use.
5. Peptide according to claim 4 for use in the treatment of skin conditions which are ameliorated by an increase of the amount of collagen.
6. The peptide according to any one of claims 1 to 5 for use in promoting wound healing and in the treatment of burns, skin ulcers, chronic wounds, bedsores.
7. The peptide according to any one of claims 4 to 6 wherein A is selected from acetyl or palmitoyl.
8. The peptide according to any one of claims 4 to 6 selected in the group consisting of:
- Acetyl-Pro-His-Arg-Glu-Asn-Lys-(N8-Palmitoyl);
- Acetyl-Pro-His-Arg-Glu-Gln;
- Acetyl-Pro-His-Arg-Glu;
- His-Arg-Glu-Asn;
- Acetyl-Pro-Arg-Glu-Asn;
- Acetyl-Hyp-His-Arg-Glu-Asn;
- Acetyl-Pro-His-Glu-Asn;
- Acetyl-Pro-His-Arg-Glu-Asn (SEQ ID No. 4); - Palm-Pro-His-Arg-Glu-Asn;
9. Use of the peptide according to any one of previous claims to prevent and/or reduce the cutaneous signs of aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress.
10. A composition comprising at least one peptide according to anyone of claims 1 to 8 and a dermatologically acceptable vehicle.
11. The composition according to claim 10 wherein said peptide is present at a concentration between 0.0005% w/w and 0.5% w/w.
12. The composition according to claims 10 or 1 1 further comprising at least one additional ingredient selected from the group comprising vitamin C, derivatives of vitamin C, vitamin E, derivatives of vitamin E, tocopherols, vitamin A, derivatives of vitamin A, retinal, retinoic acid.
13. The composition according claim 12 further containing at least one other active ingredient .
14. The composition according claim 13 wherein the at least one other active ingredient is selected from the group consisting of: an anti-radical agent or antioxidant.
15. The composition according to any one of claims 10 to 14 in the form of a cream, emulsion, dispersion, gel, ointment or lotion.
16. The composition according to any one of claims 10 to 15 for use in treatment of skin conditions which are ameliorated by an increase of the amount of collagen.
17. Use of the composition according to anyone of claims from 10 to 15 to prevent and/or reduce the cutaneous signs of aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress.
18. A method for the treatment and/or prevention of a skin condition that is ameliorated by an increase of the amount and/or stability of collagen comprising at least one step that consists in applying, to the skin or scalp, at least one peptide as defined in any one of claim 1 to 8 or at least one composition as defined in any one of claim 10 to 15.
19. A method for the treatment and/or prevention of the skin in order to combat the signs of skin aging and/or photoaging of the skin and/or to prevent and/or treat skin disorders induced by an oxidative stress, comprising at least one step that consists in applying, to the skin or scalp, at least one peptide as defined in any one of claim 1 to 8 or at least one composition as defined in any one of claim 10 to 15.
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CN114478695A (en) * 2021-11-25 2022-05-13 暨南大学 Novel polypeptide for promoting tissue repair and application thereof
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US10034826B2 (en) 2014-12-23 2018-07-31 Avon Products, Inc. Peptides and their use in the treatment of skin
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US10668046B2 (en) 2017-12-18 2020-06-02 Ansella Therapeutics, Inc. Compositions and methods for preventing and treating conditions
WO2021029684A1 (en) * 2019-08-14 2021-02-18 주식회사 에스알바이오텍 Peptide-complexed microneedle and cosmetic composition comprising same
EP4045003A4 (en) * 2019-10-18 2023-11-22 Deciem Beauty Group Inc. Skin care formulation with lipophilic peptides
CN114478695A (en) * 2021-11-25 2022-05-13 暨南大学 Novel polypeptide for promoting tissue repair and application thereof
CN114478695B (en) * 2021-11-25 2024-01-23 暨南大学 New polypeptide for promoting tissue repair and application thereof

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